NORMAL GENOMIC

• Barthe P, Mukamolova GV, Roumestand C, Cohen-Gonsaud M
• The structure of PknB extracellular PASTA domain from mycobacteriumtuberculosis suggests a ligand-dependent kinase activation.
• Structure. 2010; 18: 606-15
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• PknB is a transmembrane Ser/Thr protein kinase that defines and belongs toan ultraconserved kinase subfamily found in Gram-positive bacteria.Essential for Mycobacterium tuberculosis growth, its close homolog inBacillus subtilis has been linked to exit from dormancy. The kinasepossesses an extracellular region composed of a repetition of PASTAdomains, believed to bind peptidoglycan fragments that might act as asignaling molecule. We report here the first solution structure of thisextracellular region. Small-angle X-ray scattering and nuclear magneticresonance studies show that the four PASTA domains display an unexpectedlinear organization, contrary to what is observed in the distant proteinPBP2x from Streptococccus pneumoniae where two PASTA domains fold over ina compact structure. We propose a model for PknB activation based on aligand-dependent dimerization of the extracellular PASTA domains thatinitiates multiple signaling pathways.

• Zervosen A, Lu WP, Chen Z, White RE, Demuth TP Jr, Frere JM
• Interactions between penicillin-binding proteins (PBPs) and two novelclasses of PBP inhibitors, arylalkylidene rhodanines and arylalkylideneiminothiazolidin-4-ones.
• Antimicrob Agents Chemother. 2004; 48: 961-9
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• Several non-beta-lactam compounds were active against variousgram-positive and gram-negative bacterial strains. The MICs ofarylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-ones werelower than those of ampicillin and cefotaxime for methicillin-resistantStaphylococcus aureus MI339 and vancomycin-resistant Enterococcus faeciumEF12. Several compounds were found to inhibit the cell wall synthesis ofS. aureus and the last two steps of peptidoglycan biosynthesis catalyzedby ether-treated cells of Escherichia coli or cell wall membranepreparations of Bacillus megaterium. The effects of the arylalkylidenerhodanines and arylalkylidene iminothiazolidin-4-one derivatives on E.coli PBP 3 and PBP 5, Streptococcus pneumoniae PBP 2xS (PBP 2x from apenicillin-sensitive strain) and PBP 2xR (PBP 2x from apenicillin-resistant strain), low-affinity PBP 2a of S. aureus, and theActinomadura sp. strain R39 and Streptomyces sp. strain R61 DD-peptidaseswere studied. Some of the compounds exhibited inhibitory activities in the10 to 100 microM concentration range. The inhibition of PBP 2xS by severalof them appeared to be noncompetitive. The dissociation constant for thebest inhibitor (Ki = 10 microM) was not influenced by the presence of thesubstrate.

• Melckebeke HV et al.
• Solution structural study of BlaI: implications for the repression ofgenes involved in beta-lactam antibiotic resistance.
• J Mol Biol. 2003; 333: 711-20
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• beta-Lactamase and penicillin-binding protein PBP2' mediate staphylococcalresistance to beta-lactam antibiotics, which are otherwise highlyclinically effective. Two repressors (BlaI and MecI) regulate expressionof these inducible proteins. Here, we present the first solution structureof the 82 amino acid residue DNA-binding domain of Bacillus licheniformisBlaI which is very similar in primary sequence to the medicallysignificant Staphyloccocal BlaI and MecI proteins. This structure iscomposed of a compact core of three alpha-helices and a three-strandedbeta-sheet typical of the winged helix protein (WHP) family. Theprotein/DNA complex was studied by NMR chemical shift comparison betweenthe free and complexed forms of BlaI. Residues involved in DNA interactionwere identified and a WHP canonical model of interaction with theoperators is proposed. In this model, specific contacts occur between thebase-pairs of the TACA motif and conserved amino acid residues of therepressor helix H3. These results help toward understanding the repressionand induction mechanism of the genes coding for beta-lactamase and PBP2'.

• Katz AH, Caufield CE
• Structure-based design approaches to cell wall biosynthesis inhibitors.
• Curr Pharm Des. 2003; 9: 857-66
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• This review summarizes some of the published attempts to incorporateprotein and NMR structures in the design of new antibiotics thatspecifically target Cell Wall biosynthesis. Most of the steps involved inpeptidglycan synthesis have been investigated as potential strategiesagainst cell wall inhibition. Structural information has been most usefulin the design of molecules in the Mur enzyme pathway, penicillin bindingproteins and lactamases, as well as proteins that are part of the finalsteps of transglycosylation - in particular, d-Ala-d-Ala ligase. Severalunique issues exist in the design of effective antibacterials, such as thesignificant differences in protein structure between organisms, such asthe case of MurB in which a large amino acid loop that occupies the activesite of the E. Coli is gone in the Staph aureus enzyme. Additionally,bacterial resistance is an important issue, and in some cases, structuralinformation can be used to understand the source of this resistance. Forexample, mutations within the d-Ala-d-Ala ligases lead to the inability ofVancomycin antibiotics to bind.

• Santos JM, Lobo M, Matos AP, De Pedro MA, Arraiano CM
• The gene bolA regulates dacA (PBP5), dacC (PBP6) and ampC (AmpC),promoting normal morphology in Escherichia coli.
• Mol Microbiol. 2002; 45: 1729-40
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• The gene bolA has been shown to trigger the formation of osmoticallystable round cells when overexpressed in stationary phase. We show that inpoor growth conditions bolA is essential for normal cell morphology instationary phase and under conditions of starvation. During exponentialgrowth bolA promotes round morphology through a mechanism that is strictlydependent on the two main Escherichia colid,d-carboxypeptidases, PBP5 andPBP6. The results show that bolA controls the levels of transcription ofdacA (PBP5), dacC (PBP6) and ampC (AmpC), a class C beta-lactamase, thusconnecting for the first time penicillin binding proteins (PBPs) andbeta-lactamases at the level of gene regulation. Furthermore, PBP5 andPBP6 are shown to be independently regulated and to have distinct effectson the peptidoglycan layer. The evidence presented demonstrates that bolAis a regulator of cell wall biosynthetic enzymes with different roles incell morphology and cell division.

• Mainardi JL et al.
• Balance between two transpeptidation mechanisms determines the expressionof beta-lactam resistance in Enterococcus faecium.
• J Biol Chem. 2002; 277: 35801-7
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• The d,d-transpeptidase activity of high molecular weightpenicillin-binding proteins (PBPs) is essential to maintain cell wallintegrity as it catalyzes the final cross-linking step of bacterialpeptidoglycan synthesis. We investigated a novel beta-lactam resistancemechanism involving by-pass of the essential PBPs by l,d-transpeptidationin Enterococcus faecium. Determination of the peptidoglycan structure byreverse phase high performance liquid chromatography coupled to massspectrometry revealed that stepwise selection for ampicillin resistanceled to the gradual replacement of the usual cross-links generated by thePBPs (d-Ala(4) --> d-Asx-Lys(3)) by cross-links resulting froml,d-transpeptidation (l-Lys(3) --> d-Asx-Lys(3)). This was associated withno modification of the level of production of the PBPs or of theiraffinity for beta-lactams, indicating that altered PBP activity was notrequired for ampicillin resistance. A beta-lactam-insensitivel,d-transpeptidase was detected in membrane preparations of the parentalsusceptible strain. Acquisition of resistance was not because of variationof this activity. Instead, selection led to production of abeta-lactam-insensitive d,d-carboxypeptidase that cleaved the C-terminald-Ala residue of pentapeptide stems in vitro and caused massiveaccumulation of cytoplasmic precursors containing a tetrapeptide stem invivo. The parallel dramatic increase in the proportion of l-Lys(3) -->d-Asx-Lys(3) cross-links showed that the enzyme was activating theresistance pathway by generating the substrate for the l,d-transpeptidase.

• Ghuysen JM, Goffin C
• Lack of cell wall peptidoglycan versus penicillin sensitivity: newinsights into the chlamydial anomaly.
• Antimicrob Agents Chemother. 1999; 43: 2339-44

• Henze UU, Berger-Bachi B
• Staphylococcus aureus penicillin-binding protein 4 and intrinsicbeta-lactam resistance.
• Antimicrob Agents Chemother. 1995; 39: 2415-22
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• Increased levels of production of penicillin-binding protein PBP 4correlated with in vitro acquired intrinsic beta-lactam resistance in amutant derived from a susceptible strain of Staphylococcus aureus, strainSG511 Berlin. Truncation of the PBP 4 C-terminal membrane anchor abolishedthe PBP 4 content of cell membrane preparations as well as the resistancephenotype. A single nucleotide change and a 90-nucleotide deletion,comprising a 14-nucleotide inverted repeat in the noncoding pbp4 genepromoter proximal region, were the only sequence differences between theresistant mutant and the susceptible parent. These mutations were thoughtto be responsible for the observed overproduction of PBP 4 in theintrinsically beta-lactam-resistant mutant. The pbp4 gene was flankedupstream by the open reading frame abcA, coding for an ATP-bindingcassette transporter-like protein showing similarities to eukaryoticmultidrug transporters and downstream by a glycerol 3-phosphatecytidyltransferase (tagD)-like open reading frame presumably involved inteichoic acid synthesis. The abcA-pbp4-tagD gene cluster was located inthe SmaI-D fragment in the S. aureus 8325 chromosome in close proximity tothe RNA polymerase gene rpoB.

• Kuzin AP, Liu H, Kelly JA, Knox JR
• Binding of cephalothin and cefotaxime to D-ala-D-ala-peptidase reveals afunctional basis of a natural mutation in a low-affinitypenicillin-binding protein and in extended-spectrum beta-lactamases.
• Biochemistry. 1995; 34: 9532-40
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• Two clinically-important beta-lactam antibiotics, cephalothin andcefotaxime, have been observed by X-ray crystallography bound to thereactive Ser62 of the D-alanyl-D-alanine carboxypeptidase/transpeptidaseof Streptomyces sp. R61. Refinement of the two crystal structures producedR factors for 3 sigma (F) data of 0.166 (to 1.8 A) and 0.170 (to 2.0 A)for the cephalothin and cefotaxime complexes, respectively. In eachcomplex, a water molecule is within 3.1 and 3.6 A of the acylatedbeta-lactam carbonyl carbon atom, but is poorly activated by active siteresidues for nucleophilic attack and deacylation. This apparent lack ofgood stereochemistry for facile hydrolysis is in accord with the longhalf-lives of cephalosporin intermediates in solution (20-40 h) and theefficacy of these beta-lactams as inhibitors of bacterial cell wallsynthesis. Different hydrogen binding patterns of the two cephalosporinsto Thr301 are consistent with the low cefotaxime affinity of an alteredpenicillin-binding protein, PBP-2x, reported in cefotaxime-resistantstrains of Streptococcus pneumoniae, and with the ability of mutant classA beta-lactamases to hydrolyze third-generation cephalosporins.

• Miller JR, Ingolia TD
• Cloning and characterization of beta-lactam biosynthetic genes.
• Mol Microbiol. 1989; 3: 689-95
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• Seven genes coding for two different enzymes of thepenicillin/cephalosporin biosynthetic pathway have been cloned from fungaland bacterial sources. Using amino acid sequences derived from thepurified enzymes, oligonucleotide probes were designed to hybridize totheir cognate genes in a genomic library. The high degree of similarity(57%) between enzymes of bacterial and fungal origin suggests a horizontaltransfer of a primordial beta-lactam pathway, probably from a bacterialcell to a fungal cell. Overproduction of the proteins in Escherichia colihas allowed further study of the mechanism of action of these importantenzymes.

• Tuomanen E, Cozens R, Tosch W, Zak O, Tomasz A
• The rate of killing of Escherichia coli by beta-lactam antibiotics isstrictly proportional to the rate of bacterial growth.
• J Gen Microbiol. 1986; 132: 1297-304
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• Nongrowing bacteria evade the bactericidal activity of beta-lactamantibiotics. We sought to determine if slow growth rate also altersbactericidal activity. The bactericidal activity of two beta-lactams onEscherichia coli grown in glucose limited chemostats was compared forgeneration times ranging from 0.7 to 12 h. The degree of killing variedwith drug structure and with E. coli strain. However, all killing rateswere a constant function of the bacterial generation time: slowly growingbacteria became progressively more phenotypically tolerant to beta-lactamantibiotics as the generation time was extended.

• Then RL, Angehrn P
• Ways to overcome cephalosporinase-mediated beta-lactam resistance inEnterobacter cloacae.
• Chemioterapia. 1985; 4: 83-9
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• Beta-lactam antibiotics which affect mainly PBP 3 and are poor substratesfor the cephalosporinase of Enterobacter cloacae, but tightly bind to it,are not active against cephalosporinase-overproducing variants of thisorganism. Attempts have been made to greatly reduce the affinity for thebeta-lactamase in order to prevent both binding and hydrolysis.3-Quaternary ammonium cephalosporins and some cephalosporin (S)-sulfoxideswere seen to fit this requirement. Suitable substitution of the monocyclicbeta-lactam nucleus also resulted in compounds with lower affinity thanaztreonam. Temocillin, differing from ticarcillin by the 6-methoxy group,also has a lower affinity for the cephalosporinase. All compoundsdiscussed retain the highest affinity for PBP 3 and are active againstcephalosporinase-overproducing E. cloacae in contrast to compounds withhigh affinity for the cephalosporinase.

• Rojo F et al.
• Binding of 125I-labeled beta-lactam antibiotics to the penicillin bindingproteins of Escherichia coli.
• J Antibiot (Tokyo). 1984; 37: 389-93
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• 125I-Labeled derivatives of the beta-lactam antibiotics cephalexin,cephradine, cefaclor and 6-alpha-aminopenicillanic acid have been obtainedby reacting these compounds with (125I)-Bolton-Hunter reagent. Thefollowing target proteins were found in Escherichia coli: (1) Thederivatives of cephalexin, cefaclor and cephradine preferentially interactwith the high molecular weight penicillin binding proteins ( PBP1a andPBP1b ); (2) The 125I- derivative of 6-alpha-aminopenicillanic acid ispreferentially bound by the low molecular weight penicillin bindingproteins 4 and 5/6. The iodinated derivatives showed a very high affinityof binding to their target proteins with apparent half-saturatingconcentrations in the nano -molar range.