Secondary literature sources for FolB

The following references were automatically generated.

Jia M et al.
A D-psicose 3-epimerase with neutral pH optimum from Clostridium bolteae for D-psicose production: cloning, expression, purification, and characterization.
Appl Microbiol Biotechnol. 2014; 98: 717-25
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D-Tagatose 3-epimerase family enzymes can efficiently catalyze the epimerization of free keto-sugars, which could be used for D-psicose production from D-fructose. In previous studies, all optimum pH values of these enzymes were found to be alkaline. In this study, a D-psicose 3-epimerase (DPEase) with neutral pH optimum from Clostridium bolteae (ATCC BAA-613) was identified and characterized. The gene encoding the recombinant DPEase was cloned and expressed in Escherichia coli. In order to characterize the catalytic properties, the recombinant DPEase was purified to electrophoretic homogeneity using nickel-affinity chromatography. Ethylenediaminetetraacetic acid was shown to inhibit the enzyme activity completely; therefore, the enzyme was identified as a metalloprotein that exhibited the highest activity in the presence of Co(2)(+). Although the DPEase demonstrated the most activity at a pH ranging from 6.5 to 7.5, it exhibited optimal activity at pH 7.0. The optimal temperature for the recombinant DPEase was 55 degrees C, and the half-life was 156 min at 55 degrees C. Using D-psicose as the substrate, the apparent K(m), k(cat), and catalytic efficiency (k(cat)/K(m)) were 27.4 mM, 49 s(-)(1), and 1.78 s(-)(1) mM(-)(1), respectively. Under the optimal conditions, the equilibrium ratio of D-fructose to D-psicose was 69:31. For high production of D-psicose, 216 g/L D-psicose could be produced with 28.8 % turnover yield at pH 6.5 and 55 degrees C. The recombinant DPEase exhibited weak-acid stability and thermostability and had a high affinity and turnover for the substrate D-fructose, indicating that the enzyme was a potential D-psicose producer for industrial production.

Gu L, Zhang J, Liu B, Wu C, Du G, Chen J
High-level extracellular production of D-Psicose-3-epimerase with recombinant Escherichia coli by a two-stage glycerol feeding approach.
Bioprocess Biosyst Eng. 2013; 36: 1767-77
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The aim of this study is to achieve high-level extracellular production of D-Psicose-3-epimerase (DPE) with recombinant Escherichia coli. High-level production of DPE is one of the key factors in D-Psicose production. In the present study, the gene AAL45544.1 from Agrobacterium tumefaciens str. C58 was modified by artificial synthesis for overexpression in E. coli. The total DPE activity reached 3.96 U mL(-1) after optimization of the media composition, induction temperature, and concentration of inducer. Furthermore, it was found that addition of glycine had a positive effect on the extracellular production of DPE, which reached 3.5 U mL(-1). Finally, a two-stage glycerol feeding strategy based on both the specific growth rate before induction and the amount of glycerol residues after induction was applied in a 3-L fermenter. After a series of optimal strategies in the 3-L fermenter, the total and extracellular DPE activity were 5.08- and 3.11-fold higher than that noted in the shake flask. The extracellular and intracellular DPE activity reached 10.9 and 13.2 U mL(-1), achieving 25.5 and 31.1 % conversion of D-fructose to D-psicose, respectively. The systemic strategies presented in this study provide valuable novel information for the industrial application of DPE.

You ZY, Liu ZQ, Zheng YG, Shen YC
Characterization and application of a newly synthesized 2-deoxyribose-5-phosphate aldolase.
J Ind Microbiol Biotechnol. 2013; 40: 29-39
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A codon-optimized 2-deoxyribose-5-phosphate aldolase (DERA) gene was newly synthesized and expressed in Escherichia coli to investigate its biochemical properties and applications in synthesis of statin intermediates. The expressed DERA was purified and characterized using 2-deoxyribose-5-phosphate as the substrate. The specific activity of recombinant DERA was 1.8 U/mg. The optimum pH and temperature for DERA activity were pH 7.0 and 35 degrees C, respectively. The recombinant DERA was stable at pH 4.0-7.0 and at temperatures below 50 degrees C. The enzyme activity was inhibited by 1 mM of Ni(2+), Ba(2+) and Fe(2+). The apparent K (m) and V (max) values of purified enzyme for 2-deoxyribose-5-phosphate were 0.038 mM and 2.9 mumol min(-1) mg(-1), for 2-deoxyribose were 0.033 mM and 2.59 mumol min(-1) mg(-1), respectively, which revealed that the enzyme had similar catalytic efficiency towards phosphorylated and non-phosphorylated substrates. To synthesize statin intermediates, the bioconversion process for production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose from chloroacetaldehyde and acetaldehyde by the recombinant DERA was developed and a conversion of 94.4 % was achieved. This recombinant DERA could be a potential candidate for application in production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose.

Sola-Carvajal A, Sanchez-Carron G, Garcia-Garcia MI, Garcia-Carmona F, Sanchez-Ferrer A
Properties of BoAGE2, a second N-acetyl-D-glucosamine 2-epimerase from Bacteroides ovatus ATCC 8483.
Biochimie. 2012; 94: 222-30
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N-Acyl-D-Glucosamine 2-epimerase (AGE) catalyzes the reversible epimerization between N-acetyl-D-mannosamine (ManNAc) and N-acetyl-D-glucosamine (GlcNAc). Bacteroides ovatus ATCC 8483 shows 3 putative genes for AGE activity (BACOVA_00274, BACOVA_01795 and BACOVA_01816). The BACOVA_00274 gene encodes an AGE (BoAGE1) with strong similarity to the AGE previously characterized in Bacteroides fragilis. Interestingly, the BACOVA_01816 gene (BoAGE2) shares 57% identity with Anabaena sp. CH1 AGE, but has an extra 27-amino acid tag sequence in the N-terminal. When cloned and expressed in Escherichia coli Rosetta (DE3)pLys, BACOVA_01816 was able to convert ManNAc into GlcNAc and vice versa. It was stable over a broad range of pHs and its activity was enhanced by ATP (20 muM). The incubation with ATP stabilized its structure, raising its melting temperature by about 8 degrees C. In addition, the catalytic efficiency for ManNAc synthesis was higher than that for GlcNAc synthesis. These characteristics make BoAGE2 a promising biocatalyst for sialic acid production using cheap GlcNAc as starting material. BoAGE2 could be considered a Renin-binding Protein and its interaction with renin was studied for the first time in a prokaryotic AGE. Surprisingly, renin activated BoAGE2, an effect which is contrary to that described for mammalian AGE and unrelated with the unique N-terminal tag, since a mutant without this tag was also activated by renin. When BoAGE2 sequence was compared with other related (real and putative) AGE described in the databases, it was seen that AGE enzymes can be divided in 3 different groups. The relationship between these groups is also discussed.

Czekster CM, Blanchard JS
One substrate, five products: reactions catalyzed by the dihydroneopterin aldolase from Mycobacterium tuberculosis.
J Am Chem Soc. 2012; 134: 19758-71
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Tetrahydrofolate cofactors are required for one carbon transfer reaction involved in the synthesis of purines, amino acids, and thymidine. Inhibition of tetrahydrofolate biosynthesis is a powerful therapeutic strategy in the treatment of several diseases, and the possibility of using antifolates to inhibit enzymes from Mycobacterium tuberculosis has been explored. This work focuses on the study of the first enzyme in tetrahydrofolate biosynthesis that is unique to bacteria, dihydroneopterin aldolase (MtDHNA). This enzyme requires no metals or cofactors and does not form a protein-mediated Schiff base with the substrate, unlike most aldolases. Here, we were able to demonstrate that the reaction catalyzed by MtDHNA generates three different pterin products, one of which is not produced by other wild-type DHNAs. The enzyme-substrate complex partitions 51% in the first turnover to form the aldolase products, 24% to the epimerase product and 25% to the oxygenase products. The aldolase reaction is strongly pH dependent, and apparent pK(a) values were obtained for the first time for this class of enzyme. Furthermore, chemistry is rate limiting for the aldolase reaction, and the analysis of solvent kinetic isotope effects in steady-state and pre-steady-state conditions, combined with proton inventory studies, revealed that two protons and a likely solvent contribution are involved in formation and breakage of a common intermediate. This study provides information about the plasticity required from a catalyst that possesses high substrate specificity while being capable of utilizing two distinct epimers with the same efficiency to generate five distinct products.

Park CS, Kim JE, Choi JG, Oh DK
Characterization of a recombinant cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus and its application in the production of mannose from glucose.
Appl Microbiol Biotechnol. 2011; 92: 1187-96
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A putative N-acyl-D-glucosamine 2-epimerase from Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was identified as a cellobiose 2-epimerase by the analysis of the activity for substrates, acid-hydrolyzed products, and amino acid sequence. The cellobiose 2-epimerase was purified with a specific activity of 35 nmol min(-1) mg(-1) for D-glucose with a 47-kDa monomer. The epimerization activity for D-glucose was maximal at pH 7.5 and 75 degrees C. The half-lives of the enzyme at 60 degrees C, 65 degrees C, 70 degrees C, 75 degrees C, and 80 degrees C were 142, 71, 35, 18, and 4.6 h, respectively. The enzyme catalyzed the epimerization reactions of the aldoses harboring hydroxyl groups oriented in the right-hand configuration at the C2 position and the left-hand configuration at the C3 position, such as D-glucose, D-xylose, L-altrose, L-idose, and L-arabinose, to their C2 epimers, such as D-mannose, D-lyxose, L-allose, L-gulose, and L-ribose, respectively. The enzyme catalyzed also the isomerization reactions. The enzyme exhibited the highest activity for mannose among monosaccharides. Thus, mannose at 75 g l(-1) and fructose at 47.5 g l(-1) were produced from 500 g l(-1) glucose at pH 7.5 and 75 degrees C over 3 h by the enzyme.

Bhaskar V, Kumar M, Manicka S, Tripathi S, Venkatraman A, Krishnaswamy S
Identification of biochemical and putative biological role of a xenolog from Escherichia coli using structural analysis.
Proteins. 2011; 79: 1132-42
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YagE is a 33 kDa prophage protein encoded by CP4-6 prophage element in Escherichia coli K12 genome. Here, we report the structures of YagE complexes with pyruvate (PDB Id 3N2X) and KDGal (2-keto-3-deoxy galactonate) (PDB Id 3NEV) at 2.2A resolution. Pyruvate depletion assay in presence of glyceraldehyde shows that YagE catalyses the aldol condensation of pyruvate and glyceraldehyde. Our results indicate that the biochemical function of YagE is that of a 2-keto-3-deoxy gluconate (KDG) aldolase. Interestingly, E. coli K12 genome lacks an intrinsic KDG aldolase. Moreover, the over-expression of YagE increases cell viability in the presence of certain bactericidal antibiotics, indicating a putative biological role of YagE as a prophage encoded virulence factor enabling the survival of bacteria in the presence of certain antibiotics. The analysis implies a possible mechanism of antibiotic resistance conferred by the over-expression of prophage encoded YagE to E. coli.

Royer SF, Haslett L, Crennell SJ, Hough DW, Danson MJ, Bull SD
Structurally informed site-directed mutagenesis of a stereochemically promiscuous aldolase to afford stereochemically complementary biocatalysts.
J Am Chem Soc. 2010; 132: 11753-8
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2-Keto-3-deoxygluconate aldolase from the hyperthermophile Sulfolobus solfataricus is a highly thermostable type I aldolase that can catalyze carbon-carbon bond formation using nonphosphorylated substrates. However, it exhibits poor diastereocontrol in many of its aldol reactions, including the reaction of its natural substrates, pyruvate and D-glyceraldehyde, which afford a 55:45 mixture of D-2-keto-3-deoxygluconate (D-KDGlu) and D-2-keto-3-deoxy-galactonate (D-KDGal). We have employed detailed X-ray crystallographic structural information of this aldolase bound to these diastereoisomeric aldol products to selectively target specific amino acids for mutation for the rapid creation of stereochemically complementary mutants that catalyze either (Re)- or (Si)-facial selective aldol reactions to afford either D-KDGlu or D-KDGal with good levels of diastereocontrol.

Ito S et al.
Site-directed mutagenesis of possible catalytic residues of cellobiose 2-epimerase from Ruminococcus albus.
Biotechnol Lett. 2009; 31: 1065-71
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The cellobiose 2-epimerase from Ruminococcus albus (RaCE) catalyzes the epimerization of cellobiose and lactose to 4-O-beta-D-glucopyranosyl-D-mannose and 4-O-beta-D-galactopyranosyl-D-mannose (epilactose). Based on the sequence alignment with N-acetyl-D-glucosamine 2-epimerases of known structure and on a homology-modeled structure of RaCE, we performed site-directed mutagenesis of possible catalytic residues in the enzyme, and the mutants were expressed in Escherichia coli cells. We found that R52, H243, E246, W249, W304, E308, and H374 were absolutely required for the activity of RaCE. F114 and W303 also contributed to catalysis. These residues protruded into the active-site cleft in the model (alpha/alpha)(6) core barrel structure.

Kim YM, Chang YH, Choi NS, Kim Y, Song JJ, Kim JS
Cloning, expression, and characterization of a new deoxyribose 5-phosphate aldolase from Yersinia sp. EA015.
Protein Expr Purif. 2009; 68: 196-200
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A new deoC gene encoding deoxyribose 5-phosphate aldolase (DERA) was identified in Yersinia sp. EA015 isolated from soil. The DERA gene had an open reading frame (ORF) of 672 base pairs encoding 223 amino acids to yield a protein of molecular mass 24.8 kDa. The amino acid sequence was 94% identical to that of DERA from Yersinia intermedia ATCC 29909. DERA was over-expressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific activity was 137 micromol/min/mg. The Michaelis constant (k(m) value) of DERA was 9.1 mM. DERA was optimally active at pH 6.0 and 50 degrees C. DERA was tolerant to a high concentration (300 mM) of acetaldehyde.

McCarty RM, Somogyi A, Bandarian V
Escherichia coli QueD is a 6-carboxy-5,6,7,8-tetrahydropterin synthase.
Biochemistry. 2009; 48: 2301-3
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To elucidate the early steps required during biosynthesis of a broad class of 7-deazapurine-containing natural products, we have studied the reaction catalyzed by Escherichia coli QueD, a 6-pyruvoyl-5,6,7,8-tetrahydropterin synthase (PTPS) homologue possibly involved in queuosine biosynthesis. While mammalian PTPS homologues convert 7,8-dihydroneopterin triphosphate (H(2)NTP) to 6-pyruvoyltetrahydropterin (PPH(4)) in biopterin biosynthesis, E. coli QueD catalyzes the conversion of H(2)NTP to 6-carboxy-5,6,7,8-tetrahydropterin (CPH(4)). E. coli QueD can also convert PPH(4) and sepiapterin to CPH(4), allowing a mechanism to be proposed.

Mercer AC, Meier JL, Hur GH, Smith AR, Burkart MD
Antibiotic evaluation and in vivo analysis of alkynyl Coenzyme A antimetabolites in Escherichia coli.
Bioorg Med Chem Lett. 2008; 18: 5991-4
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Pantothenamides have been the subject of much study as potential inhibitors of CoA and carrier protein dependent biosynthetic pathways. Based on an initial observation of growth inhibition in Escherichia coli by 3, we have synthesized a small panel of pantetheine analogues and re-examined the inhibitory properties of this class of antibiotics with an emphasis on understanding the ability of these compounds to act as substrates of native CoA and carrier protein utilizing biosynthetic pathways. Our findings suggest that a secondary structure-activity relationship is an important factor in the antibiotic activity of these compounds.

Taguchi H et al.
Cloning and sequencing of the gene for cellobiose 2-epimerase from a ruminal strain of Eubacterium cellulosolvens.
FEMS Microbiol Lett. 2008; 287: 34-40
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Cellobiose 2-epimerase (CE; EC 5.1.3.11) is known to catalyze the reversible epimerization of cellobiose to 4-O-beta-D-glucopyranosyl-D-mannose in Ruminococcus albus cells. Here, we report a CE in a ruminal strain of Eubacterium cellulosolvens for the first time. The nucleotide sequence of the CE had an ORF of 1218 bp (405 amino acids; 46 963.3 Da). The CE from E. cellulosolvens showed 44-54% identity to N-acyl-D-glucosamine 2-epimerase-like hypothetical proteins in the genomes of Coprococcus eutactus, Faecalibacterium prausnitzii, Clostridium phytofermentans, Caldicellulosiruptor saccharolyticus, and Eubacterium siraeum. Surprisingly, it exhibited only 46% identity to a CE from R. albus. The recombinant enzyme expressed in Escherichia coli was purified by two-step chromatography. The purified enzyme had a molecular mass of 46.7 kDa and exhibited optimal activity at around 35 degrees C and pH 7.0-8.5. In addition to cello-oligosaccharides, it converted lactose to epilactose (4-O-beta-D-galactopyranosyl-D-mannose).

Hadi T, Dahl U, Mayer C, Tanner ME
Mechanistic studies on N-acetylmuramic acid 6-phosphate hydrolase (MurQ): an etherase involved in peptidoglycan recycling.
Biochemistry. 2008; 47: 11547-58
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Peptidoglycan recycling is a process in which bacteria import cell wall degradation products and incorporate them back into either peptidoglycan biosynthesis or basic metabolic pathways. The enzyme MurQ is an N-acetylmuramic acid 6-phosphate (MurNAc 6-phosphate) hydrolase (or etherase) that hydrolyzes the lactyl side chain from MurNAc 6-phosphate and generates GlcNAc 6-phosphate. This study supports a mechanism involving the syn elimination of lactate to give an alpha,beta-unsaturated aldehyde with (E)-stereochemistry, followed by the syn addition of water to give product. The observation of both a kinetic isotope effect slowing the reaction of [2-(2)H]MurNAc 6-phosphate and the incorporation of solvent-derived deuterium into C2 of the product indicates that the C2-H bond is cleaved during catalysis. The observation that the solvent-derived (18)O isotope is incorporated into the C3 position of the product, but not the C1 position, provides evidence of the cleavage of the C3-O bond and argues against imine formation. The finding that 3-chloro-3-deoxy-GlcNAc 6-phosphate serves as an alternate substrate is also consistent with an elimination-addition mechanism. Upon extended incubations of MurQ with GlcNAc 6-phosphate, the alpha,beta-unsaturated aldehydic intermediate accumulates in solution, and (1)H NMR analysis indicates it exists as the ring-closed form of the (E)-alkene. A structural model is developed for the Escherichia coli MurQ and is compared to that of the structural homologue glucosamine-6-phosphate synthase. Putative active site acid/base residues are probed by mutagenesis, and Glu83 and Glu114 are found to be crucial for catalysis. The Glu83Ala mutant is essentially inactive as an etherase yet is capable of exchanging the C2 proton of substrate with solvent-derived deuterium. This suggests that Glu83 may function as the acidic residue that protonates the departing lactate.

Rea D, Fulop V, Bugg TD, Roper DI
Structure and mechanism of HpcH: a metal ion dependent class II aldolase from the homoprotocatechuate degradation pathway of Escherichia coli.
J Mol Biol. 2007; 373: 866-76
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Microorganisms are adept at degrading chemically resistant aromatic compounds. One of the longest and most well characterized aromatic catabolic pathways is the 4-hydroxyphenylacetic acid degradation pathway of Escherichia coli. The final step involves the conversion of 4-hydroxy-2-oxo-heptane-1,7-dioate into pyruvate and succinic semialdehyde. This reaction is catalyzed by 4-hydroxy-2-oxo-heptane-1,7-dioate aldolase (HpcH), a member of the divalent metal ion dependent class II aldolase enzymes that have great biosynthetic potential. We have solved the crystal structure of HpcH in the apo form, and with magnesium and the substrate analogue oxamate bound, to 1.6 A and 2.0 A, respectively. Comparison with similar structures of the homologous 2-dehydro-3-deoxygalactarate aldolase, coupled with site-directed mutagenesis data, implicate histidine 45 and arginine 70 as key catalytic residues.

Blaszczyk J, Li Y, Gan J, Yan H, Ji X
Structural basis for the aldolase and epimerase activities of Staphylococcus aureus dihydroneopterin aldolase.
J Mol Biol. 2007; 368: 161-9
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Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) and the epimerization of DHNP to 7,8-dihydromonopterin (DHMP). Although crystal structures of the enzyme from several microorganisms have been reported, no structural information is available about the critical interactions between DHNA and the trihydroxypropyl moiety of the substrate, which undergoes bond cleavage and formation. Here, we present the structures of Staphylococcus aureus DHNA (SaDHNA) in complex with neopterin (NP, an analog of DHNP) and with monapterin (MP, an analog of DHMP), filling the gap in the structural analysis of the enzyme. In combination with previously reported SaDHNA structures in its ligand-free form (PDB entry 1DHN) and in complex with HP (PDB entry 2DHN), four snapshots for the catalytic center assembly along the reaction pathway can be derived, advancing our knowledge about the molecular mechanism of SaDHNA-catalyzed reactions. An additional step appears to be necessary for the epimerization of DHMP to DHNP. Three active site residues (E22, K100, and Y54) function coordinately during catalysis: together, they organize the catalytic center assembly, and individually, each plays a central role at different stages of the catalytic cycle.

Hilditch S, Berghall S, Kalkkinen N, Penttila M, Richard P
The missing link in the fungal D-galacturonate pathway: identification of the L-threo-3-deoxy-hexulosonate aldolase.
J Biol Chem. 2007; 282: 26195-201
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The fungal path for the catabolism of D-galacturonate is only partially known. It is however distinctly different to the well-known bacterial path. The known elements of the fungal path are D-galacturonate reductase converting D-galacturonate to L-galactonate and L-galactonate dehydratase converting L-galactonate to L-threo-3-deoxy-hexulosonate (2-keto-3-deoxy-L-galactonate). Here we describe the missing link in this pathway, an aldolase converting L-threo-3-deoxy-hexulosonate to pyruvate and L-glyceraldehyde. Fungal enzymes converting L-glyceraldehyde to glycerol have been described previously. The L-threo-3-deoxy-hexulosonate aldolase activity was induced in the mold Hypocrea jecorina (Trichoderma reesei) during growth on D-galacturonate. The enzyme was purified from this mold and a partial amino acid sequence obtained. This sequence was then used to identify the corresponding gene from the H. jecorina genome. The deletion of the gene resulted in a strain unable to grow on d-galacturonate and accumulating L-threo-3-deoxy-hexulosonate. The open reading frame was cloned from cDNA and functionally expressed in the yeast Saccharomyces cerevisiae. A histidine-tagged protein was expressed, purified, and characterized. The enzyme catalyzed reaction was reversible. With L-threo-3-deoxy-hexulosonate as substrate the K(m) was 3.5 mM and with pyruvate and L-glyceraldehyde the K(m) were 0.5 and 1.2 mM, respectively.

Wang W, Seah SY
Purification and biochemical characterization of a pyruvate-specific class II aldolase, HpaI.
Biochemistry. 2005; 44: 9447-55
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HpaI, a class II pyruvate-specific aldolase involved in the catabolic pathway of hydroxyphenylacetate, is overexpressed and purified. A previous suggestion that phosphate is involved in proton transfer of pyruvate, based on the crystal structure of the homologous 2-dehydro-3-deoxygalactarate aldolase, is not substantiated from biochemical studies with HpaI. Thus, specific activities of the enzyme for the substrate 4-hydroxy-2-ketopentanoate in sodium HEPES and Tris-acetate buffers are higher than in sodium phosphate buffer. The enzyme also catalyzed the partial reaction of pyruvate proton exchange with an initial rate of 0.77 mmol min(-)(1) mg(-)(1) in phosphate-free buffer, as monitored by nuclear magnetic resonance. Steady-state kinetic analysis shows that the enzyme is also able to catalyze the aldol cleavage of 4-hydroxy-2-ketohexanoate and 3-deoxy-d-manno-oct-2-ulosonic acid (KDO). The enzyme exhibits significant oxaloacetate decarboxylase activity, with a k(cat) value 2.4-fold higher than the corresponding value for the aldol cleavage of 4-hydroxy-2-ketopentanoate. Sodium oxalate, an analogue of the enolate intermediate of the enzyme-catalyzed reaction, is a competitive inhibitor of the enzyme, with a K(i) value of 5.5 microM. Replacement of an active site arginine residue (R70) with alanine by site-specific mutagenesis resulted in an enzyme that lacks both aldolase and decarboxylase activities. The mutant enzyme is also unable to catalyze pyruvate proton exchange. The dissociation constant for pyruvate in the R70A mutant, determined by fluorescence titration, is similar to that of the wild-type enzyme, indicating that pyruvate binding is not affected by this mutation. Together, the results show that R70 influences catalysis in HpaI, particularly at the pyruvate proton exchange step.

Iliades P, Walker DJ, Castelli L, Satchell J, Meshnick SR, Macreadie IG
Cloning of the Pneumocystis jirovecii trifunctional FAS gene and complementation of its DHPS activity in Escherichia coli.
Fungal Genet Biol. 2004; 41: 1053-62
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Pneumocystis pneumonia or PCP is caused by Pneumocystis jirovecii, an obligate parasite of the human lung. In this study P. jirovecii genomic sequence encoding FAS, a trifunctional protein including dihydroneopterin aldolase (DHNA), hydroxymethyldihydropterin pyrophosphokinase (PPPK) and dihydropteroate synthase (DHPS) were identified by PCR amplification from fixed broncheolar lavage samples from patients having Pneumocystis pneumonia. The P. jirovecii trifunctional DHNA-PPPK-DHPS genes (PjFAS) showed a high degree of conservation with the rat Pneumocystis carinii and P. carinii f. sp. macaca sequences. To test the functionality of the PjFAS sequences introns were removed followed by cloning and expression of PjFAS sequences in a DHPS-disrupted Escherichia coli strain. Complementation depended on the presence of N-terminal FAS sequences in addition to a glutathione S- transferase tag to the N-terminus of PjFAS. Functional complementation allowed evaluation of DHPS mutations implicated with sulfa drug resistance.

Lokanath NK et al.
Structure of aldolase from Thermus thermophilus HB8 showing the contribution of oligomeric state to thermostability.
Acta Crystallogr D Biol Crystallogr. 2004; 60: 1816-23
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2-Deoxyribose-5-phosphate aldolase catalyzes a reversible aldol condensation of two aldehydes via formation of a covalent Schiff-base intermediate at the active lysine residue. The crystal structure of 2-deoxyribose-5-phosphate aldolase from Thermus thermophilus HB8 has been determined with and without the substrate at atomic resolution. This enzyme, which has a unique homotetramer structure, has been compared with the previously reported crystal structures of two orthologues from Escherichia coli and Aeropyrum pernix. In contrast to the similar alpha/beta-barrel fold of the monomers, substantial quaternary structural differences are observed between these three enzymes. Further comparison of the subunit-subunit interface areas of these aldolases showed a clear positive correlation between the interface area and the living temperature of the source organism. From these results, it is concluded that the oligomeric state of 2-deoxyribose-5-phosphate aldolase is important for the thermostability and not for the catalytic function.

Takahashi S, Takahashi T, Kera Y, Matsunaga R, Shibuya H, Yamada RH
Cloning and expression in Escherichia coli of the D-aspartate oxidase gene from the yeast Cryptococcus humicola and characterization of the recombinant enzyme.
J Biochem. 2004; 135: 533-40
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The D-aspartate oxidase (DDO) from the yeast Cryptococcus humicola UJ1 (ChDDO) is highly specific to D-aspartate. The gene encoding ChDDO was cloned and expressed in Escherichia coli. Sequence analysis of the ChDDO gene showed that an open reading frame of 1,110 bp interrupted by two introns encodes a protein of 370 amino acids. The deduced amino acid sequence showed an FAD-binding motif and a peroxisomal targeting signal 1 in the N-terminal region and at the C-terminus, respectively, and also the presence of certain catalytically important amino acid residues corresponding to those catalytically important in D-amino acid oxidase (DAO). The sequence exhibited only a moderate identity to human (27.4%) and bovine (28.0%) DDOs, and a rather higher identity to yeast and fungal DAOs (30.4-33.2%). Similarly, phylogenetic analysis showed that ChDDO is more closely related to yeast and fungal DAOs than to mammalian DDOs. The gene expression was regulated at the transcriptional level and specifically induced by the presence of D-aspartate as the sole nitrogen source. ChDDO was expressed in an active form in E. coli to an approximately 5-fold greater extent than in yeast. The purified recombinant enzyme was identical to the native enzyme in physicochemical and catalytic properties.

Ghosh J, Postle K
Evidence for dynamic clustering of carboxy-terminal aromatic amino acids in TonB-dependent energy transduction.
Mol Microbiol. 2004; 51: 203-13
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Escherichia coli uses the proton motive force of the cytoplasmic membrane and TonB protein to energize the active transport of iron-siderophores and vitamin B12 across the outer membrane. TonB shuttles between the cytoplasmic and outer membranes, presumably during the course of energy transduction. Previous results indicated that the carboxy-terminal 65 amino acids of TonB are essential for both its outer membrane association and activity. A highly conserved region (residues 199-216) within this domain, predicted to be an amphipathic alpha-helix, was the initial focus of this study. Scanning mutagenesis indicated that only the aromatic residues F202, W213 and Y215 were individually important for activity. When the crystal structure of a dimeric TonB carboxy-terminus subsequently became available, we observed that two additional aromatic residues outside that region, F180 and F230, were potentially engaged in end-on hydrophobic interactions with the three residues identified previously. Changing these five aromatic residues individually to alanine reduced TonB activity. Surprisingly, however, each substitution exhibited a unique phenotypic profile with respect to ability to support [55Fe]-ferrichrome transport, sensitivity to colicins B, D, Ia and M or sensitivity to bacteriophage phi80. The phenotypic results suggested that the carboxy-terminus of TonB was a flexible and dynamic domain that could interact specifically with different ligands or transporters, perhaps through the aromatic residues. The possibility of interactions among all the aromatic residues was tested using double-mutant cycle analysis. All possible combinations of alanine substitutions were constructed, with the result that TonB containing any double-alanine substitution was inactive in the phenotypic assays, while retaining the ability to associate with the outer membrane. This synergistic, rather than additive, effect of the double mutants suggested that, consistent with the flexibility suggested by analysis of the single substitutions, all the aromatic residues might be capable of interacting with one another. A means of reconciling these results with the crystal structure is presented.

Li J, Wu J, Fleischhacker AS, Woodard RW
Conversion of aquifex aeolicus 3-deoxy-d-manno-octulosonate 8-phosphate synthase, a metalloenzyme, into a nonmetalloenzyme.
J Am Chem Soc. 2004; 126: 7448-9
Display abstract
The Aquifex aeolicus 3-deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS), a class II metalloenzyme, is converted into an active nonmetalloenzyme by a single amino acid mutation, namely, C11N. The result may provide insight into the evolutionary link between the two KDO8PS classes as well as the potential role of the metal and/or asparagine in the catalytic mechanism.

Madec E et al.
Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC, a Ser/Thr kinase from Bacillus subtilis.
J Mol Biol. 2003; 330: 459-72
Display abstract
We have shown recently that PrkC, which is involved in developmental processes in Bacillus subtilis, is a Ser/Thr kinase with features of the receptor kinase family of eukaryotic Hanks kinases. In this study, we expressed and purified from Escherichia coli the cytoplasmic domain of PrkC containing the kinase and a short juxtamembrane region. This fragment, which we designate PrkCc, undergoes autophosphorylation in E.coli. PrkCc is further autophosphorylated in vitro, apparently through a trans-kinase, intermolecular reaction. PrkC also displays kinase activity with myelin basic protein. Using high mass accuracy electrospray tandem mass spectrometry (LC-MS/MS) and nanoelectrospray tandem mass spectrometry, we identified seven phosphorylated threonine and one serine residue in PrkCc. All the corresponding residues were replaced by systematic site-directed mutagenesis and the purified mutant proteins were tested for in vitro kinase activity. Single and multiple replacement of four threonine residues, clustered between residues 162 and 167 in a putative activation loop, substantially reduced kinase activity and the effect was clearly additive. Replacement of the other three threonine residues, clustered between residues 290 and 320, had relatively little effect on activity. In contrast, substitution of Ser214, which is conserved in closely related receptor kinase-like bacterial proteins, independently affected activity and may represent a novel regulatory mechanism. When projected onto a 3D structure of PrkC modelled on the structure of known Hanks kinases, the first cluster of phospho-threonine residues falls precisely in the activation loop, controlling the access of substrate and ATP to the catalytic site of many eukaryotic receptor kinases, whereas the second cluster is located in the juxtamembrane region. These results indicate that regulation of PrkC kinase activity (and presumably autophosphorylation) includes a conserved activation loop mechanism. The juxtamembrane phospho-threonine residues may be essential, for example for the recruitment of other proteins necessary for a PrkC signalling cascade or for coupling to other signalling pathways. This is the first structure-function analysis of a bacterial receptor-like kinase of the Hanks family.

Kroemer M, Merkel I, Schulz GE
Structure and catalytic mechanism of L-rhamnulose-1-phosphate aldolase.
Biochemistry. 2003; 42: 10560-8
Display abstract
The structure of L-rhamnulose-1-phosphate aldolase has been established at 1.35 A resolution in a crystal form that was obtained by a surface mutation and has one subunit of the C(4)-symmetric tetramer in the asymmetric unit. It confirms an earlier 2.7 A resolution structure which was determined in a complicated crystal form with 20 subunits per asymmetric unit. The chain fold and the active center are similar to those of L-fuculose-1-phosphate aldolase and L-ribulose-5-phosphate 4-epimerase. The active center similarity is supported by a structural comparison of all three enzymes and by the binding mode of the inhibitor phosphoglycolohydroxamate at the site of the product dihydroxyacetone phosphate for the two aldolases. The sensitivity of the catalytic rate to several mutations and a comparison with the established mechanism of the related aldolase give rise to a putative catalytic mechanism. This mechanism involves the same binding mode of the second product L-lactaldehyde in both aldolases, except for a 180 degrees flip of the aldehyde group distinguishing between the two epimers rhamnulose and fuculose. The N-terminal domain exhibits a correlated anisotropic mobility that channels the isotropic Brownian motion into a directed movement of the catalytic base and the substrate phosphate on the N-domain toward the zinc ion and the lactaldehyde on the C-terminal domain. We suggest that this movement supports the catalysis mechanically.

Gilep AA, Estabrook RW, Usanov SA
Molecular cloning and heterologous expression in E. coli of cytochrome P45017alpha. Comparison of structural and functional properties of substrate-specific cytochromes P450 from different species.
Biochemistry (Mosc). 2003; 68: 86-98
Display abstract
To elucidate the nature of substrate specificity and intrinsic mechanism of hydroxylation of steroids, in the present work we carried out molecular cloning and heterologous expression of cDNA for three new forms of cytochrome P45017alpha from species of the Bovidae family (sheep, goat, and bison), which catalyze 17alpha-hydroxylation of both progesterone (P4) or pregnenolone (P5) and 17,20-lyase reaction resulting in cleavage of side chain with formation of C(19)-steroids. Recombinant cytochromes P45017alpha were expressed in E. coli as derivatives, containing a six-His tag at the C-terminal sequence that simplifies purification of the cloned heme proteins using metal-affinity chromatography. Highly purified cytochromes P45017alpha were used for determination of enzyme activity and specificity in relation to progesterone, pregnenolone, 17alpha-hydroxyprogesterone, and 17alpha-hydroxypregnenolone with registration of the kinetics of reaction product formation using HPLC. It is shown that each form of cytochrome P45017alpha is characterized by a specific profile of enzyme activity and dependence of 17,20-lyase reaction on the presence of cytochrome b(5) in the reaction mixture. The analysis of the activity of the known forms of cytochrome P45017alpha in view of the data obtained in the present work allows the division of known cytochromes P45017alpha into three main group: group A (pig, hamster, rat), cytochromes P45017alpha catalyze the reaction of 17alpha-hydroxylation of both P4 and P5 steroids and the 17,20-lyase reaction of 17alpha-hydroxyprogesterone and 17alpha-hydroxypregnenolone; group B (human, bovine, sheep, goat, and bison), cytochromes P45017alpha, which have no or have insignificant 17,20-lyase activity in relation to 17alpha-hydroxyprogesterone; group C (guinea pig), cytochrome P45017alpha which either has no or has insignificant 17,20-lyase activity on transformation 17alpha-hydroxypregnenolone to dehydroepiandrosterone.

Daigle DM, Rossi L, Berghuis AM, Aravind L, Koonin EV, Brown ED
YjeQ, an essential, conserved, uncharacterized protein from Escherichia coli, is an unusual GTPase with circularly permuted G-motifs and marked burst kinetics.
Biochemistry. 2002; 41: 11109-17
Display abstract
The Escherichia coli protein YjeQ represents a protein family whose members are broadly conserved in bacteria and have been shown to be indispensable to the growth of E. coli and Bacillus subtilis [Arigoni, F., et al. (1998) Nat. Biotechnol. 16, 851]. Proteins of the YjeQ family contain all sequence motifs typical of the vast class of P-loop-containing GTPases, but show a circular permutation, with a G4-G1-G3 pattern of motifs as opposed to the regular G1-G3-G4 pattern seen in most GTPases. All YjeQ family proteins display a unique domain architecture, which includes a predicted N-terminal OB-fold RNA-binding domain, the central permuted GTPase module, and a zinc knuckle-like C-terminal cysteine cluster. This domain architecture suggests a possible role for YjeQ as a regulator of translation. YjeQ was overexpressed, purified to homogeneity, and shown to contain 0.6 equiv of GDP. Steady state kinetic analyses indicated slow GTP hydrolysis, with a k(cat) of 9.4 h(-)(1) and a K(m) for GTP of 120 microM (k(cat)/K(m) = 21.7 M(-)(1) s(-)(1)). YjeQ also hydrolyzed other nucleoside triphosphates and deoxynucleotide triphosphates such as ATP, ITP, and CTP with specificity constants (k(cat)/K(m)) ranging from 0.2 to 1.0 M(-)(1) s(-)(1). Pre-steady state kinetic analysis of YjeQ revealed a burst of nucleotide hydrolysis for GTP described by a first-order rate constant of 100 s(-)(1) as compared to a burst rate of 0.2 s(-)(1) for ATP. In addition, a variant in the G1 motif of YjeQ (S221A) was substantially impaired for GTP hydrolysis (0.3 s(-)(1)) with a less significant impact on the steady state rate (1.8 h(-)(1)). In summary, E. coli YjeQ is an unusual, circularly permuted P-loop-containing GTPase, which catalyzes GTP hydrolysis at a rate 45 000 times greater than that of turnover.

Thorell S, Schurmann M, Sprenger GA, Schneider G
Crystal structure of decameric fructose-6-phosphate aldolase from Escherichia coli reveals inter-subunit helix swapping as a structural basis for assembly differences in the transaldolase family.
J Mol Biol. 2002; 319: 161-71
Display abstract
Fructose-6-phosphate aldolase from Escherichia coli is a member of a small enzyme subfamily (MipB/TalC family) that belongs to the class I aldolases. The three-dimensional structure of this enzyme has been determined at 1.93 A resolution by single isomorphous replacement and tenfold non-crystallographic symmetry averaging and refined to an R-factor of 19.9% (R(free) 21.3%). The subunit folds into an alpha/beta barrel, with the catalytic lysine residue on barrel strand beta 4. It is very similar in overall structure to that of bacterial and mammalian transaldolases, although more compact due to extensive deletions of additional secondary structural elements. The enzyme forms a decamer of identical subunits with point group symmetry 52. Five subunits are arranged as a pentamer, and two ring-like pentamers pack like a doughnut to form the decamer. A major interaction within the pentamer is through the C-terminal helix from one monomer, which runs across the active site of the neighbouring subunit. In classical transaldolases, this helix folds back and covers the active site of the same subunit and is involved in dimer formation. The inter-subunit helix swapping appears to be a major determinant for the formation of pentamers rather than dimers while at the same time preserving importing interactions of this helix with the active site of the enzyme. The active site lysine residue is covalently modified, by forming a carbinolamine with glyceraldehyde from the crystallisation mixture. The catalytic machinery is very similar to that of transaldolase, which together with the overall structural similarity suggests that enzymes of the MipB/TALC subfamily are evolutionary related to the transaldolase family.

Luo Y, Samuel J, Mosimann SC, Lee JE, Tanner ME, Strynadka NC
The structure of L-ribulose-5-phosphate 4-epimerase: an aldolase-like platform for epimerization.
Biochemistry. 2001; 40: 14763-71
Display abstract
The structure of L-ribulose-5-phosphate 4-epimerase from E. coli has been solved to 2.4 A resolution using X-ray diffraction data. The structure is homo-tetrameric and displays C(4) symmetry. Each subunit has a single domain comprised of a central beta-sheet flanked on either side by layers of alpha-helices. The active site is identified by the position of the catalytic zinc residue and is located at the interface between two adjacent subunits. A remarkable feature of the structure is that it shows a very close resemblance to that of L-fuculose-1-phosphate aldolase. This is consistent with the notion that both enzymes belong to a superfamily of epimerases/aldolases that catalyze carbon-carbon bond cleavage reactions via a metal-stabilized enolate intermediate. Detailed inspection of the epimerase structure, however, indicates that despite the close overall structural similarity to class II aldolases, the enzyme has evolved distinct active site features that promote its particular chemistry.

Kim J et al.
Ancestral divergence, genome diversification, and phylogeographic variation in subpopulations of sorbitol-negative, beta-glucuronidase-negative enterohemorrhagic Escherichia coli O157.
J Bacteriol. 2001; 183: 6885-97
Display abstract
The O157:H7 lineage of enterohemorrhagic Escherichia coli is a geographically disseminated complex of highly related genotypes that share common ancestry. The common clone that is found worldwide carries several markers of events in its evolution, including markers for acquisition of virulence genes and loss of physiological characteristics, such as sorbitol fermentation ability and beta-glucuronidase production. Populations of variants that are distinct with respect to motility and the sorbitol and beta-glucuronidase markers appear to have diverged at several points along the inferred evolutionary pathway. In addition to these variants, distinct subpopulations of the contemporary non-sorbitol-fermenting, beta-glucuronidase-negative O157:H7 clone were recently detected among bovine and human clinical isolates in the United States by using high-resolution genome comparison. In order to determine if these recently described subpopulations were derived from a regional or ancestral divergence event, we used octamer-based genome scanning, marker sorting, and DNA sequence analysis to examine their phylogenetic relationship to populations of non-sorbitol-fermenting, beta-glucuronidase negative O157:H7 and O157:H- strains from Australia. The inferred phylogeny is consistent with the hypothesis that subpopulations on each continent resulted from geographic spread of an ancestral divergence event and subsequent expansion of distinct subpopulations. Marker sorting and DNA sequence analyses identified sets of monophyletic markers consistent with the pattern of divergence and demonstrated that phylogeographic variation occurred through emergence of regional subclones and concentration of regional polymorphisms among distinct subpopulations. DNA sequence analysis of representative polyphyletic markers showed that genome diversity accrued through random drift and bacteriophage-mediated events.

Tripet B, Wagschal K, Lavigne P, Mant CT, Hodges RS
Effects of side-chain characteristics on stability and oligomerization state of a de novo-designed model coiled-coil: 20 amino acid substitutions in position "d".
J Mol Biol. 2000; 300: 377-402
Display abstract
We describe the de novo design and biophysical characterization of a model coiled-coil protein in which we have systematically substituted 20 different amino acid residues in the central "d" position. The model protein consists of two identical 38 residue polypeptide chains covalently linked at their N termini via a disulfide bridge. The hydrophobic core contained Val and Ile residues at positions "a" and Leu residues at positions "d". This core allowed for the formation of both two-stranded and three-stranded coiled-coils in benign buffer, depending on the substitution at position "d". The structure of each analog was analyzed by CD spectroscopy and their relative stability determined by chemical denaturation using GdnHCI (all analogs denatured from the two-stranded state). The oligomeric state(s) was determined by high-performance size-exclusion chromatography and sedimentation equilibrium analysis in benign medium. Our results showed a thermodynamic stability order (in order of decreasing stability) of: Leu, Met, Ile, Tyr, Phe, Val, Gln, Ala, Trp, Asn, His, Thr, Lys, Ser, Asp, Glu, Arg, Orn, and Gly. The Pro analog prevented coiled-coil formation. The overall stability range was 7.4 kcal/mol from the lowest to the highest analog, indicating the importance of the hydrophobic core and the dramatic effect a single substitution in the core can have upon the stability of the protein fold. In general, the side-chain contribution to the level of stability correlated with side-chain hydrophobicity. Molecular modelling studies, however, showed that packing effects could explain deviations from a direct correlation. In regards to oligomerization state, eight analogs demonstrated the ability to populate exclusively one oligomerization state in benign buffer (0.1 M KCl, 0.05 M K(2)PO(4)(pH 7)). Ile and Val (the beta-branched residues) induced the three-stranded oligomerization state, whereas Tyr, Lys, Arg, Orn, Glu and Asp induced the two-stranded state. Asn, Gln, Ser, Ala, Gly, Phe, Leu, Met and Trp analogs were indiscriminate and populated two-stranded and three-stranded states. Comparison of these results with similar substitutions in position "a" highlights the positional effects of individual residues in defining the stability and numbers of polypeptide chains occurring in a coiled-coil structure. Overall, these results in conjunction with other work now generate a relative thermodynamic stability scale for 19 naturally occurring amino acid residues in either an "a" or "d" position of a two-stranded coiled-coil. Thus, these results will aid in the de novo design of new coiled-coil structures, a better understanding of their structure/function relationships and the design of algorithms to predict the presence of coiled-coils within native protein sequences.

Fox D, Smulian AG
Mkp1 of Pneumocystis carinii associates with the yeast transcription factor Rlm1 via a mechanism independent of the activation state.
Cell Signal. 2000; 12: 381-90
Display abstract
The mitogen-activated protein (MAP) kinase Mkp1 of the fungal pathogen Pneumocystis carinii is a functional MAP kinase that complements the loss of Slt2p, the MAP kinase component of the cell integrity pathway of Saccharomyces cerevisiae, and is activated within P. carinii in response to oxidative stress. Mkp1 displays an unusual feature in that it contains a phosphorylation motif repeat (TEYMTEY) within the activation loop not present in any other fungal MAPK identified to date. Mutagenesis of the T186,Y188 phosphorylation motif within the activation domain of Mkp1 results in the loss of detectable kinase activity but still retains partial complementation function. In addition to the ability of Mkp1 to restore partial activity to the cell integrity pathway in the absence of phosphorylatable residues within the activation loop, the association of Mkp1 with a substrate of Slt2p, the transcription factor Rlm1p, can also occur in the absence of MAP kinase activation. The results of this study suggest that the presence of phosphorylatable residues within the activation loop of Mkp1 is not absolutely required for functional (complementation) activity or for the association of Mkp1 with the transcription factor Rlm1p. In contrast, the catalytic lysine of the ATP-binding domain of Mkp1 is necessary for both complementation function and interaction with Rlm1p.

McClelland M et al.
Comparison of the Escherichia coli K-12 genome with sampled genomes of a Klebsiella pneumoniae and three salmonella enterica serovars, Typhimurium, Typhi and Paratyphi.
Nucleic Acids Res. 2000; 28: 4974-86
Display abstract
The Escherichia coli K-12 genome (ECO) was compared with the sampled genomes of the sibling species Salmonella enterica serovars Typhimurium, Typhi and Paratyphi A (collectively referred to as SAL) and the genome of the close outgroup Klebsiella pneumoniae (KPN). There are at least 160 locations where sequences of >400 bp are absent from ECO but present in the genomes of all three SAL and 394 locations where sequences are present in ECO but close homologs are absent in all SAL genomes. The 394 sequences in ECO that do not occur in SAL contain 1350 (30.6%) of the 4405 ECO genes. Of these, 1165 are missing from both SAL and KPN. Most of the 1165 genes are concentrated within 28 regions of 10-40 kb, which consist almost exclusively of such genes. Among these regions were six that included previously identified cryptic phage. A hypothetical ancestral state of genomic regions that differ between ECO and SAL can be inferred in some cases by reference to the genome structure in KPN and the more distant relative Yersinia pestis. However, many changes between ECO and SAL are concentrated in regions where all four genera have a different structure. The rate of gene insertion and deletion is sufficiently high in these regions that the ancestral state of the ECO/SAL lineage cannot be inferred from the present data. The sequencing of other closely related genomes, such as S.bongori or Citrobacter, may help in this regard.

Kawata Y et al.
Cloning and over-expression of thermostable Bacillus sp. YM55-1 aspartase and site-directed mutagenesis for probing a catalytic residue.
Eur J Biochem. 2000; 267: 1847-57
Display abstract
A thermostable aspartase gene (aspB) from Bacillus sp. YM55-1 was cloned and the gene sequenced. The aspB gene (1407 bp ORF) encodes a protein with a molecular mass of 51 627 Da, consisting of 468 amino-acid residues. An amino-acid sequence comparison revealed that Bacillus YM55-1 aspartase shared 71% homology with Bacillus subtilis aspartase and 49% with Escherichia coli and Pseudomonas fluorescens aspartases. The E. coli TK237/pUCASPB strain, which was obtained by transforming E. coli TK237 (aspartase-null strain) with a vector plasmid (pUCASPB) containing the cloned aspB gene, produced a large amount of the enzyme corresponding to > 10% of the total soluble protein. The over-expressed recombinant enzyme (native molecular mass: 200 kDa) was purified effectively and rapidly using heat treatment and affinity chromatography. In order to probe the catalytic residues of this enzyme, two conserved amino-acid residues, Lys183 and His134, were individually mutated to alanine. Although the tertiary structure of each mutant was estimated to be the same as that of wild-type aspartase in CD and fluorescence measurements, the Lys183Ala mutant lost its activity completely, whereas His134Ala retained full activity. This finding suggests that Lys183 may be involved in the catalytic activity of this thermostable Bacillus YM55-1 aspartase.

Campbell RE, Mosimann SC, Tanner ME, Strynadka NC
The structure of UDP-N-acetylglucosamine 2-epimerase reveals homology to phosphoglycosyl transferases.
Biochemistry. 2000; 39: 14993-5001
Display abstract
Bacterial UDP-N-acetylglucosamine 2-epimerase catalyzes the reversible epimerization at C-2 of UDP-N-acetylglucosamine (UDP-GlcNAc) and thereby provides bacteria with UDP-N-acetylmannosamine (UDP-ManNAc), the activated donor of ManNAc residues. ManNAc is critical for several processes in bacteria, including formation of the antiphagocytic capsular polysaccharide of pathogens such as Streptococcus pneumoniae types 19F and 19A. We have determined the X-ray structure (2.5 A) of UDP-GlcNAc 2-epimerase with bound UDP and identified a previously unsuspected structural homology with the enzymes glycogen phosphorylase and T4 phage beta-glucosyltransferase. The relationship to these phosphoglycosyl transferases is very intriguing in terms of possible similarities in the catalytic mechanisms. Specifically, this observation is consistent with the proposal that the UDP-GlcNAc 2-epimerase-catalyzed elimination and re-addition of UDP to the glycal intermediate may proceed through a transition state with significant oxocarbenium ion-like character. The homodimeric epimerase is composed of two similar alpha/beta/alpha sandwich domains with the active site located in the deep cleft at the domain interface. Comparison of the multiple copies in the asymmetric unit has revealed that the epimerase can undergo a 10 degrees interdomain rotation that is implicated in the regulatory mechanism. A structure-based sequence alignment has identified several basic residues in the active site that may be involved in the proton transfer at C-2 or stabilization of the proposed oxocarbenium ion-like transition state. This insight into the structure of the bacterial epimerase is applicable to the homologous N-terminal domain of the bifunctional mammalian UDP-GlcNAc "hydrolyzing" 2-epimerase/ManNAc kinase that catalyzes the rate-determining step in the sialic acid biosynthetic pathway.

Rosano C et al.
Probing the catalytic mechanism of GDP-4-keto-6-deoxy-d-mannose Epimerase/Reductase by kinetic and crystallographic characterization of site-specific mutants.
J Mol Biol. 2000; 303: 77-91
Display abstract
GDP-4-keto-6-deoxy-d-mannose epimerase/reductase is a bifunctional enzyme responsible for the last step in the biosynthesis of GDP-l-fucose, the substrate of fucosyl transferases. Several cell-surface antigens, including the leukocyte Lewis system and cell-surface antigens in pathogenic bacteria, depend on the availability of GDP-l-fucose for their expression. Therefore, the enzyme is a potential target for therapy in pathological states depending on selectin-mediated cell-to-cell interactions. Previous crystallographic investigations have shown that GDP-4-keto-6-deoxy-d-mannose epimerase/reductase belongs to the short-chain dehydrogenase/reductase protein homology family. The enzyme active-site region is at the interface of an N-terminal NADPH-binding domain and a C-terminal domain, held to bind the substrate. The design, expression and functional characterization of seven site-specific mutant forms of GDP-4-keto-6-deoxy-d-mannose epimerase/reductase are reported here. In parallel, the crystal structures of the native holoenzyme and of three mutants (Ser107Ala, Tyr136Glu and Lys140Arg) have been investigated and refined at 1. 45-1.60 A resolution, based on synchrotron data (R-factors range between 12.6 % and 13.9 %). The refined protein models show that besides the active-site residues Ser107, Tyr136 and Lys140, whose mutations impair the overall enzymatic activity and may affect the coenzyme binding mode, side-chains capable of proton exchange, located around the expected substrate (GDP-4-keto-6-deoxy-d-mannose) binding pocket, are selectively required during the epimerization and reduction steps. Among these, Cys109 and His179 may play a primary role in proton exchange between the enzyme and the epimerization catalytic intermediates. Finally, the additional role of mutated active-site residues involved in substrate recognition and in enzyme stability has been analyzed.

Joerger AC, Gosse C, Fessner WD, Schulz GE
Catalytic action of fuculose 1-phosphate aldolase (class II) as derived from structure-directed mutagenesis.
Biochemistry. 2000; 39: 6033-41
Display abstract
Previous analyses established the structures of unligated L-fuculose 1-phosphate aldolase and of the enzyme ligated with an inhibitor mimicking the substrate dihydroxyacetone phosphate. These data allowed us to suggest a catalytic mechanism. On the basis of this proposal, numerous mutations were now introduced at the active center and tested with respect to their catalytic rates and their product distributions. For several mutants, the structures were determined. The results demonstrate the catalytic importance of some particular residues in defined conformations and in the mobile C-terminal chain end. Moreover, they led to a modification of the proposed mechanism. The effect of some mutations on enantioselectivity and on the ratio of diastereomer formation indicates clearly the binding site of the aldehyde moiety in relation to the other substrate dihydroxyacetone phosphate.

Zhang S, Wilson DB, Ganem B
Probing the catalytic mechanism of prephenate dehydratase by site-directed mutagenesis of the Escherichia coli P-protein dehydratase domain.
Biochemistry. 2000; 39: 4722-8
Display abstract
The Escherichia coli bifunctional P-protein, which plays a central role in L-phenylalanine (Phe) biosynthesis, contains distinct chorismate mutase (CM) and prephenate dehydratase (PDT) domains as well as a regulatory (R) domain for feedback control by Phe. To elucidate the catalytic mechanism of PDT in the P-protein, 24 mutations of 15 conserved residues in the PDT domain were created, expressed in the pheA(-)E. coli strain NK6024, and studied for their effect on PDT activity. Fourteen mutant enzymes were purified to homogeneity, tested for feedback inhibition by Phe, and characterized by kinetic analysis and circular dichroism spectroscopy. Selected mutant enzymes were further studied by gel filtration, fluorescence emission, and microcalorimetry. In addition, a monofunctional PDT domain (PDT20, residues 101-285) was cloned and overexpressed in plasmid pET with expression levels up to 200-250 mg/L. PDT20 retained full PDT activity, lacked CM activity, and was insensitive to feedback inhibition by Phe. Four residues (T278, N160, Q215, and S208) were shown to be important for PDT catalysis. The values of k(cat)/K(m) for the S208A/C and T278S mutant enzymes were 100-fold lower, and 500-fold lower for the N160A and Q215A mutant enzymes than the wild-type (WT) protein. The T278A and T278V mutant enzymes displayed no measurable catalytic activity, yet bound both prephenate and a competitive inhibitor (S-DNBA) comparably to the WT protein. These data, taken together with the normal CD spectra of the mutant enzymes, strongly suggested that T278 was involved in the catalytic mechanism. To establish whether acidic residues were involved in catalysis, all the conserved Glu and Asp residues in the PDT domain were mutated to Ala. None of these mutations significantly reduced PDT activity, indicating that the acidic residues of the PDT domain are not directly involved in catalysis. However, two mutant enzymes (E159A and E232A) displayed higher levels of PDT activity (2.2- and 3.5-fold, respectively), which was due to enhanced substrate binding. For the double mutant enzyme (E159A-E232A), k(cat)/K(m) was ca. 7-fold higher than for the WT enzyme, while its K(m) was 4.6-fold lower.

Karaivanova IM et al.
Mutational analysis of the thermostable arginine repressor from Bacillus stearothermophilus: dissecting residues involved in DNA binding properties.
J Mol Biol. 1999; 291: 843-55
Display abstract
Recently the crystal structure of the DNA-unbound form of the full-length hexameric Bacillus stearothermophilus arginine repressor (ArgR) has been resolved, providing a possible explanation for the mechanism of arginine-mediated repressor-operator DNA recognition. In this study we tested some of these functional predictions by performing site-directed mutagenesis of distinct amino acid residues located in two regions, the N-terminal DNA-binding domain and the C-terminal oligomerization domain of ArgR. A total of 15 mutants were probed for their capacity to repress the expression of the reporter argC - lacZ gene fusion in Escherichia coli cells. Substitutions of highly conserved amino acid residues in the alpha2 and alpha3 helices, located in the winged helix-turn-helix DNA-binding motif, reduced repression. Loss of DNA-binding capacity was confirmed in vitro for the Ser42Pro mutant which showed the most pronounced effect in vivo. In E. coli, the wild-type B. stearothermophilus ArgR molecule behaves as a super-repressor, since recombinant E. coli host cells bearing B. stearothermophilusargR on a multicopy vector did not grow in selective minimal medium devoid of arginine and grew, albeit weakly, when l -arginine was supplied. All mutants affected in the DNA-binding domain lost this super-repressor behaviour. Replacements of conserved leucine residues at positions 87 and/or 94 in the C-terminal domain by other hydrophobic amino acid residues proved neutral or caused either derepression or stronger super-repression. Substitution of Leu87 by phenylalanine was found to increase the DNA-binding affinity and the protein solubility in the context of a double Leu87Phe/Leu94Val mutant. Structural modifications occasioned by the various amino acid substitutions were confirmed by circular dichroism analysis and structure modelling.

Thompson J, Pikis A, Ruvinov SB, Henrissat B, Yamamoto H, Sekiguchi J
The gene glvA of Bacillus subtilis 168 encodes a metal-requiring, NAD(H)-dependent 6-phospho-alpha-glucosidase. Assignment to family 4 of the glycosylhydrolase superfamily.
J Biol Chem. 1998; 273: 27347-56
Display abstract
The gene glvA (formerly glv-1) from Bacillus subtilis has been cloned and expressed in Escherichia coli. The purified protein GlvA (449 residues, Mr = 50,513) is a unique 6-phosphoryl-O-alpha-D-glucopyranosyl:phosphoglucohydrolase (6-phospho-alpha-glucosidase) that requires both NAD(H) and divalent metal (Mn2+, Fe2+, Co2+, or Ni2+) for activity. 6-Phospho-alpha-glucosidase (EC 3.2.1.122) from B. subtilis cross-reacts with polyclonal antibody to maltose 6-phosphate hydrolase from Fusobacterium mortiferum, and the two proteins exhibit amino acid sequence identity of 73%. Estimates for the Mr of GlvA determined by SDS-polyacrylamide gel electrophoresis (51,000) and electrospray-mass spectroscopy (50,510) were in excellent agreement with the molecular weight of 50,513 deduced from the amino acid sequence. The sequence of the first 37 residues from the N terminus determined by automated analysis agreed precisely with that predicted by translation of glvA. The chromogenic and fluorogenic substrates, p-nitrophenyl-alpha-D-glucopyranoside 6-phosphate and 4-methylumbelliferyl-alpha-D-glucopyranoside 6-phosphate were used for the discontinuous assay and in situ detection of enzyme activity, respectively. Site-directed mutagenesis shows that three acidic residues, Asp41, Glu111, and Glu359, are required for GlvA activity. Asp41 is located at the C terminus of a betaalphabeta fold that may constitute the dinucleotide binding domain of the protein. Glu111 and Glu359 may function as the catalytic acid (proton donor) and nucleophile (base), respectively, during hydrolysis of 6-phospho-alpha-glucoside substrates including maltose 6-phosphate and trehalose 6-phosphate. In metal-free buffer, GlvA exists as an inactive dimer, but in the presence of Mn2+ ion, these species associate to form the NAD(H)-dependent catalytically active tetramer. By comparative sequence alignment with its homologs, the novel 6-phospho-alpha-glucosidase from B. subtilis can be assigned to the nine-member family 4 of the glycosylhydrolase superfamily.

Bornemann S, Lowe DJ, Thorneley RN
Escherichia coli chorismate synthase.
Biochem Soc Trans. 1996; 24: 84-8

Maru I, Ohta Y, Murata K, Tsukada Y
Molecular cloning and identification of N-acyl-D-glucosamine 2-epimerase from porcine kidney as a renin-binding protein.
J Biol Chem. 1996; 271: 16294-9
Display abstract
N-Acetylneuraminic acid (NeuAc) is an important molecule in biological recognition systems. NeuAc is known to be biosynthesized either from UDP-N-acetyl-D-glucosamine by an action of UDP-N-acetyl-D-glucosamine 2-epimerase or from N-acetyl-D-glucosamine by N-acyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase). However, the physiological function of the GlcNAc 2-epimerase in NeuAc biosynthesis has not been fully evaluated. To clarify the role of GlcNAc 2-epimerase in NeuAc biosynthesis, the enzyme and its gene were isolated from porcine kidney cortex. Escherichia coli cells transformed with the gene expressed the GlcNAc 2-epimerase having the same properties as those of the GlcNAc 2-epimerase from porcine kidney. Sequence analysis indicated that the gene was capable of synthesizing a 46.5-kDa protein (402 amino acids) with a conserved leucine zipper motif. Homology search for the cloned gene revealed that the GlcNAc 2-epimerase was identical with renin-binding protein (RnBP) in porcine kidney (Inoue, H., Fukui, K., Takahashi, S., and Miyake, Y.(1990) J. Biol. Chem. 265, 6556-6561) (identity: 99.6% in nucleotide sequence, 99.0% in amino acid sequence). That GlcNAc 2-epimerase is a RnBP was confirmed by its ability to bind porcine kidney renin and mask its protease activity. These findings provide unequivocal evidence that the enzyme GlcNAc 2-epimerase is a RnBP.

Murley LL, MacKenzie RE
The two monofunctional domains of octameric formiminotransferase-cyclodeaminase exist as dimers.
Biochemistry. 1995; 34: 10358-64
Display abstract
Formiminotransferase-cyclodeaminase is a bifunctional enzyme arranged as a circular tetramer of dimers that exhibits the ability to efficiently channel polyglutamylated folate between catalytic sites. Through deletion mutagenesis we demonstrate that each subunit consists of an N-terminal transferase active domain and a C-terminal deaminase active domain separated by a linker sequence of minimally eight residues. The full-length enzyme and both isolated domains have been expressed as C-terminally histidine-tagged proteins. Both domains self-dimerize, providing direct evidence for the existence of two types of subunit interfaces. The results suggest that both the transferase and the deaminase activities are dependent on the formation of specific subunit interfaces. Because channeling is not observed between isolated domains, only the octamer appears able to directly transfer pentaglutamylated intermediate between active sites.

Reichel C, Bang H, Brune K, Geisslinger G, Menzel S
2-Arylpropionyl-CoA epimerase: partial peptide sequences and tissue localization.
Biochem Pharmacol. 1995; 50: 1803-6
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The R-enantiomers of 2-arylpropionic acids (2-APAs) such as ibuprofen (IBU) exhibit the phenomenon of species- and substrate-dependent metabolic chiral inversion. Only R-enantiomers are activated to acyl-CoA-thioesters by an acyl-CoA-synthetase via an adenylate intermediate. The acyl-CoA-thioesters are substrates for an epimerase, which is responsible for chiral inversion. A 42 kDa epimerase from the cytosolic fraction of rat livers was isolated and purified to homogeneity. Polyclonal antibodies were raised against the epimerase in rabbits. The anti-epimerase antibodies were used for affinity column chromatography to separate homogeneous protein for amino acid sequence analysis. Sequence data analysis of 3 internal peptide sequences showed 50% and more homology with regions of enzymes involved in fatty acid metabolism. The polyclonal anti-epimerase antibodies were used to analyze the tissue distribution of the in guinea pigs and rats by Western blot analysis. Furthermore, the correlation of inversion enzyme activity in various tissues under comparable incubation conditions and cross-reactivity in Western blot analysis was investigated.

Taha TS, Deits TL
Purification and characterization of 2-keto-3-deoxy-6-phosphogluconate aldolase from Azotobacter vinelandii: evidence that the enzyme is bifunctional towards 2-keto-4-hydroxy glutarate cleavage.
Biochem Biophys Res Commun. 1994; 200: 459-66
Display abstract
2-keto-3-deoxy-6-phosphogluconate aldolase (E.C. 4.1.2.14) has been purified in two chromatographic steps to 99% purity in 73% overall yield from Azotobacter vinelandii. The pure enzyme is a 70 kD trimeric Class I aldolase, inhibitable by bromopyruvate or pyruvate plus sodium borohydride, with a specific activity of 625 mumol per min per mg protein and a Km of 38 microM for 2-keto-3-deoxy-6-phosphogluconate. The enzyme also has 2-keto-4-hydroxy glutarate aldolase (E.C. 4.1.3.16) activity, with a specific activity of 4.8 mumol per min per mg protein and a Km of 39 microM. 2-keto-4-hydroxy glutarate inhibits the 2-keto-3-deoxy-6-phosphogluconate aldolase activity of the enzyme with an apparent Ki of 0.17 mM. Slow steps following formation of the Schiff base intermediate between KHG and the enzyme are responsible for both the slower turnover of this substrate and for its inhibitory effect.

Smeland TE, Li J, Cuebas D, Schulz H
The mechanism and function of 3-hydroxyacyl-CoA epimerase in rat liver and Escherichia coli.
Prog Clin Biol Res. 1992; 375: 85-93

Nakamura T, Nagasawa T, Yu F, Watanabe I, Yamada H
A new catalytic function of halohydrin hydrogen-halide-lyase, synthesis of beta-hydroxynitriles from epoxides and cyanide.
Biochem Biophys Res Commun. 1991; 180: 124-30
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Halohydrin hydrogen-halide-lyase, which catalyzes the interconversion of halohydrins to epoxides, purified from a recombinant E. coli was found to catalyze the transformation of 1,2-epoxybutane into beta-hydroxyvaleronitrile in the presence of cyanide. Chloride inhibited competitively the formation of beta-hydroxyvaleronitrile. The enzyme also catalyzed the transformation of some other epoxides into the corresponding beta-hydroxynitriles in the presence of cyanide.

Hoffman M
On the road to mandelate ... racemase.
Science. 1991; 251: 31-2

Wetzler C, Behrens U
[Formation and action of epimerase from Azotobacter vinelandii].
Z Allg Mikrobiol. 1982; 22: 73-6
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After exhaustion of the C-source, terminating the accumulation of the extracellular polysaccharide, the excretion of the epimerase is increased, accordingly the moiety of guluronosyls of the alginate is augmented. Epimerization reactions with an epimerase preparation with commercial alginates from algae lead to higher conversion rates than with alginate from Azotobacter vinelandii. It is reasoned that the acetyl groups of bacterial alginate perform a steric hinderance. Because of the small amount of guluronosyls in Azotobacter alginate, these alginates do not form gels with Ca++.

Ziegler EJ, McCutchan JA, Douglas H, Braude AI
Prevention of lethal pseudomonas bacteremia with epimerase-deficient E. coli antiserum.
Trans Assoc Am Physicians. 1975; 88: 101-8
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All of these experiments demonstrate that despite many microbiologic differences between Pseudomonas and the enteric rods, there is a close immunologic relationship between their endotoxin cores. These studies also show that by genetic manipulation we can unmask a protective antigen for preparing antiserum with a broad range of activity, even in the face of neutropenia. Production of potent human J5 antiserum is a safe, simple procedure, making it possible to consider endotoxin core antibody treatment of all Gram negative bacteremia in patients regardless of the causative organism.

Fan CL, Krivi GG, Brown GM
The conversion of dihydroneopterin triphosphate to sepiapterin by an enzyme system from Drosophila melanogaster.
Biochem Biophys Res Commun. 1975; 67: 1047-54

Dainty RH
Purification and properties of threonine aldolase from Clostridium pasteurianum.
Biochem J. 1967; 104: 46-46