NORMAL GENOMIC

• Soupene E, Rothschild J, Kuypers FA, Dean D
• Eukaryotic protein recruitment into the Chlamydia inclusion: implications for survival and growth.
• PLoS One. 2012; 7: 36843-36843
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• Chlamydia trachomatis (Ct) is an obligate intracellular human pathogen that multiplies within a parasitophorous vacuole called an inclusion. We report that the location of several host-cell proteins present in the cytosol, the nucleus, and membranes was altered during Ct development. The acyl-CoA synthetase enzyme ACSL3 and the soluble acyl-CoA binding protein ACBD6 were mobilized from organelle membranes and the nucleus, respectively, into the lumen of the inclusion. The nuclear protein ZNF23, a pro-apoptosis factor, was also translocated into the inclusion lumen. ZNF23, among other proteins, might be targeted by Ct to inhibit host cell apoptosis, thereby enabling bacterial survival. In contrast, the acyl-CoA:lysophosphatidylcholine acyltransferase LPCAT1, an endoplasmic reticulum membrane protein, was recruited to the inclusion membrane. The coordinated action of ACBD6, ACSL3 and LPCAT1 likely supports remodeling and scavenging of host lipids into bacterial-specific moieties essential to Ct growth. To our knowledge, these are the first identified host proteins known to be intercepted and translocated into the inclusion.

• Roos K, Siegbahn PE
• Density functional theory study of the manganese-containing ribonucleotide reductase from Chlamydia trachomatis: why manganese is needed in the active complex.
• Biochemistry. 2009; 48: 1878-87
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• The active center of Chlamydia trachomatis (Ct) ribonucleotide reductase (RNR) has been studied using B3LYP hybrid density functional theory. Class Ic Ct RNR lacks the radical-bearing tyrosine that is crucial for activity in conventional class I (subclass a and b) RNR. Instead of the Fe(III)Fe(III)Tyr(rad) active state in conventional class I, Ct RNR has Mn(IV)Fe(III) at the metal center of subunit II. Based on calculated (H(+), e(-))-binding energies for Ct R2, iron-substituted Ct R2, and R2 from Escherichia coli (Ec), an explanation is proposed for why the enzyme needs this novel metal center. Mn(IV) is shown to be an equally strong oxidant as the tyrosyl radical in Ec R2. Fe(IV), however, is a much too strong oxidant and would therefore not be possible in the active cofactor. The structure of the catalytic center of the active state, such as protonation state and position of Mn, is discussed. Ct R2 has a different ligand structure than conventional class I R2 with a fourth Glu (like MMO) instead of three Glu and one Asp. Calculations indicate that, in the presence of Tyr, Glu at this position is less flexible than Asp, whereas with Phe both Glu and Asp are equally flexible. This may be a reason why conventional class I RNR has an Asp, while Ct R2, lacking the tyrosine, has a Glu.

• Sun X, Baker HM, Ge R, Sun H, He QY, Baker EN
• Crystal structure and metal binding properties of the lipoprotein MtsA, responsible for iron transport in Streptococcus pyogenes.
• Biochemistry. 2009; 48: 6184-90
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• An ability to acquire iron is essential for the viability and growth of almost all organisms and in pathogenic bacteria is strongly correlated with virulence. The cell surface lipoprotein MtsA, a component of the MtsABC transporter of Streptococcus pyogenes, acts as the primary receptor for inorganic iron by this significant human pathogen. Iron is bound as Fe(2+), with the participation of bicarbonate. The crystal structure of MtsA has been determined and refined at 1.8 A resolution (R = 0.167, and R(free) = 0.194). MtsA has the classic bacterial metal binding receptor (MBR) fold, with the Fe(2+) ion bound to the side chains of His68, His140, Glu206, and Asp281, at a totally enclosed site between the two domains of the protein. The absence of bicarbonate from the binding site suggests that it is displaced during the final stages of metal binding. Both the fold and metal binding site are most similar to those of the manganese receptors PsaA and MntC, consistent with the similar coordination requirements of Fe(2+) and Mn(2+). Binding studies confirm a 10-fold preference for Fe(2+) over Mn(2+), although both may be carried in vivo. Mutational analysis of the binding site shows that His140 is critical for a fully functional binding site but that Glu206 is dispensable. The crystal structure explains the distinct roles of these ligands and also reveals potential secondary binding sites that may explain the binding behavior of MtsA for metal ions other than Fe(2+).

• Kosinski J, Plotz G, Guarne A, Bujnicki JM, Friedhoff P
• The PMS2 subunit of human MutLalpha contains a metal ion binding domain of the iron-dependent repressor protein family.
• J Mol Biol. 2008; 382: 610-27
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• DNA mismatch repair (MMR) is responsible for correcting replication errors. MutLalpha, one of the main players in MMR, has been recently shown to harbor an endonuclease/metal-binding activity, which is important for its function in vivo. This endonuclease activity has been confined to the C-terminal domain of the hPMS2 subunit of the MutLalpha heterodimer. In this work, we identify a striking sequence-structure similarity of hPMS2 to the metal-binding/dimerization domain of the iron-dependent repressor protein family and present a structural model of the metal-binding domain of MutLalpha. According to our model, this domain of MutLalpha comprises at least three highly conserved sequence motifs, which are also present in most MutL homologs from bacteria that do not rely on the endonuclease activity of MutH for strand discrimination. Furthermore, based on our structural model, we predict that MutLalpha is a zinc ion binding protein and confirm this prediction by way of biochemical analysis of zinc ion binding using the full-length and C-terminal domain of MutLalpha. Finally, we demonstrate that the conserved residues of the metal ion binding domain are crucial for MMR activity of MutLalpha in vitro.

• Kim AC, Oliver DC, Paetzel M
• Crystal structure of a bacterial signal Peptide peptidase.
• J Mol Biol. 2008; 376: 352-66
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• Signal peptide peptidase (Spp) is the enzyme responsible for cleaving the remnant signal peptides left behind in the membrane following Sec-dependent protein secretion. Spp activity appears to be present in all cell types, eukaryotic, prokaryotic and archaeal. Here we report the first structure of a signal peptide peptidase, that of the Escherichia coli SppA (SppA(EC)). SppA(EC) forms a tetrameric assembly with a novel bowl-shaped architecture. The bowl has a dramatically hydrophobic interior and contains four separate active sites that utilize a Ser/Lys catalytic dyad mechanism. Our structural analysis of SppA reveals that while in many Gram-negative bacteria as well as characterized plant variants, a tandem duplication in the protein fold creates an intact active site at the interface between the repeated domains, other species, particularly Gram-positive and archaeal organisms, encode half-size, unduplicated SppA variants that could form similar oligomers to their duplicated counterparts, but using an octamer arrangement and with the catalytic residues provided by neighboring monomers. The structure reveals a similarity in the protein fold between the domains in the periplasmic Ser/Lys protease SppA and the monomers seen in the cytoplasmic Ser/His/Asp protease ClpP. We propose that SppA may, in addition to its role in signal peptide hydrolysis, have a role in the quality assurance of periplasmic and membrane-bound proteins, similar to the role that ClpP plays for cytoplasmic proteins.

• Cramer JF, Nordberg PA, Hajdu J, Lejon S
• Crystal structure of a bacterial albumin-binding domain at 1.4 A resolution.
• FEBS Lett. 2007; 581: 3178-82
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• The albumin-binding domain, or GA module, of the peptostreptococcal albumin-binding protein expressed in pathogenic strains of Finegoldia magna is believed to be responsible for the virulence and increased growth rate of these strains. Here we present the 1.4A crystal structure of this domain, and compare it with the crystal structure of the GA-albumin complex. An analysis of protein-protein interactions in the two crystals, and the presence of multimeric GA species in solution, indicate the GA module is "sticky", and is capable of forming contacts with a range of protein surfaces. This might lead to interactions with different host proteins.

• Key J, Hefti M, Purcell EB, Moffat K
• Structure of the redox sensor domain of Azotobacter vinelandii NifL at atomic resolution: signaling, dimerization, and mechanism.
• Biochemistry. 2007; 46: 3614-23
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• NifL is a multidomain sensor protein responsible for the transcriptional regulation of genes involved in response to changes in cellular redox state and ADP concentration. Cellular redox is monitored by the N-terminal PAS domain of NifL which contains an FAD cofactor. Flavin-based PAS domains of this type have also been referred to as LOV domains. To explore the mechanism of signal recognition and transduction in NifL, we determined the crystal structure of the FAD-bound PAS domain of NifL from Azotobacter vinelandii to 1.04 A resolution. The structure reveals a novel cavity within the PAS domain which contains two water molecules directly coordinated to the FAD. This cavity is connected to solvent by multiple access channels which may facilitate the oxidation of the FAD by molecular oxygen and the release of hydrogen peroxide. The structure contains a dimer of the NifL PAS domain that is structurally very similar to those described in other crystal structures of PAS domains and identifies a conserved dimerization motif. An N-terminal amphipathic helix constitutes part of the dimerization interface, and similar N-terminal helices are identified in other PAS domain proteins. The structure suggests a model for redox-mediated signaling in which a conformational change is initiated by redox-dependent changes in protonation at the N5 atom of FAD that lead to reorganization of hydrogen bonds within the flavin binding pocket. A structural signal is subsequently transmitted to the beta-sheet interface between the monomers of the PAS domain.

• Cuypers MG, Mitchell EP, Romao CV, McSweeney SM
• The crystal structure of the Dps2 from Deinococcus radiodurans reveals an unusual pore profile with a non-specific metal binding site.
• J Mol Biol. 2007; 371: 787-99
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• The crystal structure of recombinant Dps2 (DRB0092, DNA protecting protein under starved conditions) from the Gram-positive, radiation-resistant bacterium Deinococcus radiodurans has been determined in its apo and iron loaded states. Like other members of the Dps family, the bacterial DrDps2 assembles as a spherical dodecamer with an outer shell diameter of 90 A and an interior diameter of 40 A. A total of five iron sites were located in the iron loaded structure, representing the first stages of iron biomineralisation. Each subunit contains a mononuclear iron ferroxidase centre coordinated by residues highly conserved amongst the Dps family of proteins. In the structures presented, a distinct iron site is observed 6.1 A from the ferroxidase centre with a unique ligand configuration of mono coordination by the protein and no bridging ligand to the ferroxidase centre. A non-specific metallic binding site, suspected to play a regulative role in iron uptake/release from the cage, was found in a pocket located near to the external edge of the C-terminal 3-fold channel.

• Samuel T, Welsh K, Lober T, Togo SH, Zapata JM, Reed JC
• Distinct BIR domains of cIAP1 mediate binding to and ubiquitination of tumor necrosis factor receptor-associated factor 2 and second mitochondrial activator of caspases.
• J Biol Chem. 2006; 281: 1080-90
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• Inhibitor of apoptosis proteins (IAPs) regulate apoptosis primarily by inhibiting caspase-family proteases. However, many IAPs also possess E3 ligase (ubiquitin-protein isopeptide ligase) activities implicated in both caspase-dependent and -independent functions of these proteins. Here, we compared the structural features of cIAP1 responsible for its interactions with two known target proteins, TRAF2 and SMAC. The N-terminal (BIR1) and C-terminal (BIR3) BIR domains of cIAP1 were determined to be necessary and sufficient for binding TRAF2 and SMAC, respectively. Mutational analysis of the BIR1 and BIR3 domains identified critical residues required for TRAF2 and SMAC binding. Using these mutants, cIAP1-mediated ubiquitination of TRAF2 and SMAC in vitro was determined to be correspondingly dependent on intact binding sites on BIR1 and BIR3. Because TRAF2 regulates NF-kappaB activation, the effects of cIAP1 on TRAF2-mediated induction of NF-kappaB transcriptional activity were studied using reporter gene assays. Expression of a fragment of cIAP1 encompassing the three BIR domains (but not full-length cIAP1) greatly enhanced TRAF2-induced increases in NF-kappaB activity, providing a convenient assay for monitoring BIR-dependent effects of cIAP1 on TRAF2 in cells. BIR1 mutants of the BIR1-3 fragment of cIAP1 that failed to bind TRAF2 lost the ability to modulate NF-kappaB activity, demonstrating a requirement for BIR1-mediated interactions with TRAF2. Altogether, these findings demonstrate the modularity and diversification of BIR domains, showing that a single cIAP can direct its E3 ligase activity toward different substrates and can alter the cellular functions of different protein targets in accordance with differences in the specificity of individual BIR domains.

• Goellner S, Schubert E, Liebler-Tenorio E, Hotzel H, Saluz HP, Sachse K
• Transcriptional response patterns of Chlamydophila psittaci in different in vitro models of persistent infection.
• Infect Immun. 2006; 74: 4801-8
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• The obligatory intracellular bacterium Chlamydophila psittaci is the causative agent of psittacosis in birds and humans. The capability of this zoonotic pathogen to develop a persistent phase is likely to play a role in chronicity of infections, as well as in failure of antibiotic therapy and immunoprophylaxis. To elucidate three different in vitro models for transition of C. psittaci to persistence (iron depletion, penicillin G treatment, and gamma interferon [IFN-gamma] exposure), a set of 27 genes was examined by mRNA expression analysis using quantitative real-time PCR. While the phenotypical characteristics were the same as in other chlamydiae, i.e., aberrant morphology of reticulate bodies, loss of cultivability, and rescue of infectivity upon removal of inducers, the transcriptional response of C. psittaci to persistence-inducing factors included several new and distinctive features. Consistent downregulation of membrane proteins, chlamydial sigma factors, cell division protein, and reticulate body-elementary body differentiation proteins from 24 h postinfection onward proved to be a general feature of C. psittaci persistence. However, other genes displayed considerable variations in response patterns from one model to another, which suggests that there is no persistence model per se. In contrast to results for Chlamydia trachomatis, late shutdown of essential genes in C. psittaci was more comprehensive with IFN-gamma-induced persistence, which is probably due to the absence of a functional tryptophan synthesis operon.

• Amor JC et al.
• The structure of RalF, an ADP-ribosylation factor guanine nucleotide exchange factor from Legionella pneumophila, reveals the presence of a cap over the active site.
• J Biol Chem. 2005; 280: 1392-400
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• The Legionella pneumophila protein RalF is secreted into host cytosol via the Dot/Icm type IV transporter where it acts to recruit ADP-ribosylation factor (Arf) to pathogen-containing phagosomes in the establishment of a replicative organelle. The presence in RalF of the Sec7 domain, present in all Arf guanine nucleotide exchange factors, has suggested that recruitment of Arf is an early step in pathogenesis. We have determined the crystal structure of RalF and of the isolated Sec7 domain and found that RalF is made up of two domains. The Sec7 domain is homologous to mammalian Sec7 domains. The C-terminal domain forms a cap over the active site in the Sec7 domain and contains a conserved folding motif, previously observed in adaptor subunits of vesicle coat complexes. The importance of the capping domain and of the glutamate in the "glutamic finger," conserved in all Sec7 domains, to RalF functions was examined using three different assays. These data highlight the functional importance of domains other than Sec7 in Arf guanine nucleotide exchange factors to biological activities and suggest novel mechanisms of regulation of those activities.

• Ilari A et al.
• The unusual intersubunit ferroxidase center of Listeria innocua Dps is required for hydrogen peroxide detoxification but not for iron uptake. A study with site-specific mutants.
• Biochemistry. 2005; 44: 5579-87
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• The role of the ferroxidase center in iron uptake and hydrogen peroxide detoxification was investigated in Listeria innocua Dps by substituting the iron ligands His31, His43, and Asp58 with glycine or alanine residues either individually or in combination. The X-ray crystal structures of the variants reveal only small alterations in the ferroxidase center region compared to the native protein. Quenching of the protein fluorescence was exploited to assess stoichiometry and affinity of metal binding. Substitution of either His31 or His43 decreases Fe(II) affinity significantly with respect to wt L. innocua Dps (K approximately 10(5) vs approximately 10(7) M(-)(1)) but does not alter the binding stoichiometry [12 Fe(II)/dodecamer]. In the H31G-H43G and H31G-H43G-D58A variants, binding of Fe(II) does not take place with measurable affinity. Oxidation of protein-bound Fe(II) increases the binding stoichiometry to 24 Fe(III)/dodecamer. However, the extent of fluorescence quenching upon Fe(III) binding decreases, and the end point near 24 Fe(III)/dodecamer becomes less distinct with increase in the number of mutated residues. In the presence of dioxygen, the mutations have little or no effect on the kinetics of iron uptake and in the formation of micelles inside the protein shell. In contrast, in the presence of hydrogen peroxide, with increase in the number of substitutions the rate of iron oxidation and the capacity to inhibit Fenton chemistry, thereby protecting DNA from oxidative damage, appear increasingly compromised, a further indication of the role of ferroxidation in conferring peroxide tolerance to the bacterium.

• Hu J, Hubbard SR
• Structural characterization of a novel Cbl phosphotyrosine recognition motif in the APS family of adapter proteins.
• J Biol Chem. 2005; 280: 18943-9
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• The Cbl adapter proteins typically function to down-regulate activated protein tyrosine kinases and other signaling proteins by coupling them to the ubiquitination machinery for degradation by the proteasome. Cbl proteins bind to specific tyrosine-phosphorylated sequences in target proteins via the tyrosine kinase-binding (TKB) domain, which comprises a four-helix bundle, an EF-hand calcium-binding domain, and a non-conventional Src homology-2 domain. The previously derived consensus sequence for phosphotyrosine recognition by the Cbl TKB domain is NXpY(S/T)XXP (X denotes lesser residue preference), wherein specificity is conferred primarily by residues C-terminal to the phosphotyrosine. Cbl is recruited to and phosphorylated by the insulin receptor in adipose cells through the adapter protein APS. APS is phosphorylated by the insulin receptor on a C-terminal tyrosine residue, which then serves as a binding site for the Cbl TKB domain. Using x-ray crystallography, site-directed mutagenesis, and calorimetric studies, we have characterized the interaction between the Cbl TKB domain and the Cbl recruitment site in APS, which contains a sequence motif, RA(V/I)XNQpY(S/T), that is conserved in the related adapter proteins SH2-B and Lnk. These studies reveal a novel mode of phosphopeptide interaction with the Cbl TKB domain, in which N-terminal residues distal to the phosphotyrosine directly contact residues of the four-helix bundle of the TKB domain.

• Boonserm P, Davis P, Ellar DJ, Li J
• Crystal structure of the mosquito-larvicidal toxin Cry4Ba and its biological implications.
• J Mol Biol. 2005; 348: 363-82
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• Cry4Ba, isolated from Bacillus thuringiensis subsp. israelensis, is specifically toxic to the larvae of Aedes and Anopheles mosquitoes. The structure of activated Cry4Ba toxin has been determined by multiple isomorphous replacement with anomalous scattering and refined to R(cryst) = 20.5% and R(free)= 21.8% at 1.75 Angstroms resolution. It resembles previously reported Cry toxin structures but shows the following distinctions. In domain I the helix bundle contains only the long and amphipathic helices alpha3-alpha7. The N-terminal helices alpha1-alpha2b, absent due to proteolysis during crystallisation, appear inessential to toxicity. In domain II the beta-sheet prism presents short apical loops without the beta-ribbon extension of inner strands, thus placing the receptor combining sites close to the sheets. In domain III the beta-sandwich contains a helical extension from the C-terminal strand beta23, which interacts with a beta-hairpin excursion from the edge of the outer sheet. The structure provides a rational explanation of recent mutagenesis and biophysical data on this toxin. Furthermore, added to earlier structures from the Cry toxin family, Cry4Ba completes a minimal structural database covering the Coleoptera, Lepidoptera, Diptera and Lepidoptera/Diptera specificity classes. A multiple structure alignment found that the Diptera-specific Cry4Ba is structurally more closely similar to the Lepidoptera-specific Cry1Aa than the Coleoptera-specific Cry3Aa, but most distantly related to Lepidoptera/Diptera-specific Cry2Aa. The structures are most divergent in domain II, supporting the suggestion that this domain has a major role in specificity determination. They are most similar in the alpha3-alpha7 major fragment of domain I, which contains the alpha4-alpha5 hairpin crucial to pore formation. The collective knowledge of Cry toxin structure and mutagenesis data will lead to a more critical understanding of the structural basis for receptor binding and pore formation, as well as allowing the scope of diversity to be better appreciated.

• Won HS, Low LY, Guzman RD, Martinez-Yamout M, Jakob U, Dyson HJ
• The zinc-dependent redox switch domain of the chaperone Hsp33 has a novel fold.
• J Mol Biol. 2004; 341: 893-9
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• The Escherichia coli chaperone Hsp33 contains a C-terminal zinc-binding domain that modulates activity by a so-called "redox switch". The oxidized form in the absence of zinc is active, while the reduced form in the presence of zinc is inactive. X-ray crystal structures of Hsp33 invariably omit details of the C-terminal domain, which is truncated in protein constructs that are capable of forming crystals. We report the solution structure of a recombinant 61-residue protein containing the zinc-binding domain (residues 227-287) of Hsp33, in the presence of stoichiometric amounts of Zn2+. The zinc-bound protein is well folded, and forms a novel structure unlike other published zinc-binding domains. The structure consists of two helices at right-angles to each other, a two-stranded B-hairpin and a third helix at the C terminus. The zinc site comprises the side-chains of the conserved cysteine residues 232, 234, 262 and 265, and connects a short sequence before the first helix with the tight turn in the middle of the B-hairpin. The structure of the C-terminal zinc-binding domain suggests a mechanism for the operation of the redox switch: loss of the bound zinc ion disrupts the folded structure, allowing the ligand cysteine residues to be oxidized, probably to disulfide bonds. The observation that the C-terminal domain is poorly structured in the active oxidized form suggests that the loss of zinc and unfolding of the domain precedes the oxidation of the thiolate groups of the cysteine residues, since the formation of disulfides between distant parts of the domain sequence would presumably promote the formation of stable three-dimensional structure in the oxidized form.Hsp33 provides an example of a redox signaling system that utilizes protein folding and unfolding together with chemical modification for transduction of external stimuli, in this case oxidative stress, to activate the machinery of the cell that is designed to deal with that stress.

• Hogbom M, Stenmark P, Voevodskaya N, McClarty G, Graslund A, Nordlund P
• The radical site in chlamydial ribonucleotide reductase defines a new R2 subclass.
• Science. 2004; 305: 245-8
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• Ribonucleotide reductase (RNR) synthesizes the deoxyribonucleotides for DNA synthesis. The R2 protein of normal class I ribonucleotide reductases contains a diiron site that produces a stable tyrosyl free radical, essential for enzymatic activity. Structural and electron paramagnetic resonance studies of R2 from Chlamydia trachomatis reveal a protein lacking a tyrosyl radical site. Instead, the protein yields an iron-coupled radical upon reconstitution. The coordinating structure of the diiron site is similar to that of diiron oxidases/monoxygenases and supports a role for this radical in the RNR mechanism. The specific ligand pattern in the C. trachomatis R2 metal site characterizes a new group of R2 proteins that so far has been found in eight organisms, three of which are human pathogens.

• Vevodova J et al.
• Structure/function studies on a S-adenosyl-L-methionine-dependent uroporphyrinogen III C methyltransferase (SUMT), a key regulatory enzyme of tetrapyrrole biosynthesis.
• J Mol Biol. 2004; 344: 419-33
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• The crystallographic structure of the Pseudomonas denitrificans S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferase (SUMT), which is encoded by the cobA gene, has been solved by molecular replacement to 2.7A resolution. SUMT is a branchpoint enzyme that plays a key role in the biosynthesis of modified tetrapyrroles by controlling flux to compounds such as vitamin B(12) and sirohaem, and catalysing the transformation of uroporphyrinogen III into precorrin-2. The overall topology of the enzyme is similar to that of the SUMT module of sirohaem synthase (CysG) and the cobalt-precorrin-4 methyltransferase CbiF and, as with the latter structures, SUMT has the product S-adenosyl-L-homocysteine bound in the crystal. The roles of a number of residues within the SUMT structure are discussed with respect to their conservation either across the broader family of cobalamin biosynthetic methyltransferases or within the sub-group of SUMT members. The D47N, L49A, F106A, T130A, Y183A and M184A variants of SUMT were generated by mutagenesis of the cobA gene, and tested for SAM binding and enzymatic activity. Of these variants, only D47N and L49A bound the co-substrate S-adenosyl-L-methionine. Consequently, all the mutants were severely restricted in their capacity to synthesise precorrin-2, although both the D47N and L49A variants produced significant quantities of precorrin-1, the monomethylated derivative of uroporphyrinogen III. The activity of these variants is interpreted with respect to the structure of the enzyme.

• Li H, Graupner M, Xu H, White RH
• CofE catalyzes the addition of two glutamates to F420-0 in F420 coenzyme biosynthesis in Methanococcus jannaschii.
• Biochemistry. 2003; 42: 9771-8
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• The protein product of the Methanococcus jannaschii MJ0768 gene has been expressed in Escherichia coli, purified to homogeneity, and shown to catalyze the GTP-dependent addition of two l-glutamates to the l-lactyl phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F(420)-0) to form F(420)-0-glutamyl-glutamate (F(420)-2). Since the reaction is the fifth step in the biosynthesis of coenzyme F(420), the enzyme has been designated as CofE, the product of the cofE gene. Gel filtration chromatography indicates CofE is a dimer. The enzyme has no recognized sequence similarity to any previously characterized proteins. The enzyme has an absolute requirement for a divalent metal ion and a monovalent cation. Among the metal ions tested, a mixture of Mn(2+), Mg(2+), and K(+) is the most effective. CofE catalyzes amide bond formation with the cleavage of GTP to GDP and inorganic phosphate, likely involving the activation of the free carboxylate group of F(420)-0 to give an acyl phosphate intermediate. Evidence for the occurrence of this intermediate is presented. A reaction mechanism for the enzyme is proposed and compared with other members of the ADP-forming amide bond ligase family.

• Nersissian AM, Shipp EL
• Blue copper-binding domains.
• Adv Protein Chem. 2002; 60: 271-340

• Edeling MA, Guddat LW, Fabianek RA, Thony-Meyer L, Martin JL
• Structure of CcmG/DsbE at 1.14 A resolution: high-fidelity reducing activity in an indiscriminately oxidizing environment.
• Structure. 2002; 10: 973-9
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• CcmG is unlike other periplasmic thioredoxin (TRX)-like proteins in that it has a specific reducing activity in an oxidizing environment and a high fidelity of interaction. These two unusual properties are required for its role in c-type cytochrome maturation. The crystal structure of CcmG reveals a modified TRX fold with an unusually acidic active site and a groove formed from two inserts in the fold. Deletion of one of the groove-forming inserts disrupts c-type cytochrome formation. Two unique structural features of CcmG-an acidic active site and an adjacent groove-appear to be necessary to convert an indiscriminately binding scaffold, the TRX fold, into a highly specific redox protein.