Kegg Pathway: D-Alanine metabolism

BMC Microbiol. 2008; 8: 85
Kraus D, Herbert S, Kristian SA, Khosravi A, Nizet V, Götz F, Peschel A

BACKGROUND: Modification of teichoic acids with D-Alanine by the products of the dlt operon protects Gram-positive bacteria against major antimicrobial host defense molecules such as defensins, cathelicidins, myeloperoxidase or phospholipase. The graRS regulatory genes have recently been implicated in the control of D-alanylation in Staphylococcus aureus. RESULTS: To determine the impact of the GraRS regulatory system on resistance to antimicrobial host defense mechanisms and virulence of S. aureus, we compared inactivation of S. aureus SA113 wild type and its isogenic graRS deletion mutant by the human cathelicidin LL-37 or human neutrophil granulocytes in vitro, and the ability to cause infection in vivo. We show here that graRS deletion considerably alters bacterial surface charge, increases susceptibility to killing by human neutrophils or the defense peptide LL-37, and attenuates virulence of S. aureus in a mouse infection model. CONCLUSION: Our results indicate that S. aureus can regulate its surface properties in order to overcome innate host defenses.

Antibiot Khimioter. 2007; 52(6): 3-8
Orlova TI, Bulgakova VG, Polin AN, Grushina VA

Cell walls of Staphylococcus aureus R9/80 resistant to gramicidin S and actinomycin D were investigated. The strain was isolated after passages of a previously isolated strain of S. aureus with resistance to gramicidin and definite changes in the cell walls, a medium with increasing concentrations of actinomycin being used for the passages. The data on the study of the cell walls of the strain with the double resistance were compared with the results of the investigation of the cell walls of the strain susceptible to gramicidin, the gramicidin resistant strain (initial for strain R9/80) and the actinomycin adapted strain that also showed changes in the cell walls. The cell walls of the resistant strains had no significant changes in the peptidoglycane and glucosamine levels, as well as in the peptidoglycane amino acid composition. Teichoic acids of all the strains had different levels of substitution of ribite by D-Alanine (a factor influencing the negative charge of teichoic acids and the wall at large). It was noted that all the strains resistant to the tested antibiotics had lower levels of teichoic acids in the cell walls. The resistant cells showed some increase of the lipid component in the walls: from 1.6% in the susceptible strain to 2.1-2.9% in the resistant cells. The main trend of the changes in the resistance development was revealed to be the thickening of the cell wall and its consolidation. The development of resistance to gramicidin, actinomycin and to both the antibiotics provoked respectively a 2.4-, 4- and 5.4-fold increase of the content of the main cell component. i.e. peptidoglycane in the cell biomass. The barrier role of the cell walls in the resistant strains and their ability to bind the antibiotic is discussed.

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 May 1; 64(Pt 5): 327-33
Au K, Ren J, Walter TS, Harlos K, Nettleship JE, Owens RJ, Stuart DI, Esnouf RM

Bacillus anthracis, the causative agent of anthrax, has been targeted by the Oxford Protein Production Facility to validate high-throughput protocols within the Structural Proteomics in Europe project. As part of this work, the structures of an alanine racemase (BA0252) in the presence and absence of the inhibitor (R)-1-aminoethylphosphonic acid (L-Ala-P) have determined by X-ray crystallography to resolutions of 2.1 and 1.47 A, respectively. Difficulties in crystallizing this protein were overcome by the use of reductive methylation. Alanine racemase has attracted much interest as a possible target for anti-anthrax drugs: not only is D-Alanine a vital component of the bacterial cell wall, but recent studies also indicate that alanine racemase, which is accessible in the exosporium, plays a key role in inhibition of germination in B. anthracis. These structures confirm the binding mode of L-Ala-P but suggest an unexpected mechanism of inhibition of alanine racemase by this compound and could provide a basis for the design of improved alanine racemase inhibitors with potential as anti-anthrax therapies.

FEMS Microbiol Rev. 2008 Mar; 32(2): 168-207
Barreteau H, Kovac A, Boniface A, Sova M, Gobec S, Blanot D

The biosynthesis of bacterial cell wall peptidoglycan is a complex process that involves enzyme reactions that take place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner side (synthesis of lipid-linked intermediates) and outer side (polymerization reactions) of the cytoplasmic membrane. This review deals with the cytoplasmic steps of peptidoglycan biosynthesis, which can be divided into four sets of reactions that lead to the syntheses of (1) UDP-N-acetylglucosamine from fructose 6-phosphate, (2) UDP-N-acetylmuramic acid from UDP-N-acetylglucosamine, (3) UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid and (4) D-glutamic acid and dipeptide D-alanyl-D-Alanine. Recent data concerning the different enzymes involved are presented. Moreover, special attention is given to (1) the chemical and enzymatic synthesis of the nucleotide precursor substrates that are not commercially available and (2) the search for specific inhibitors that could act as antibacterial compounds.

J Microbiol Biotechnol. 2008 Jan; 18(1): 55-8
Priyadarshi A, Lee EH, Sung MW, Kim JH, Ku MJ, Kim EE, Hwang KY

Alanine racemase, a bacterial enzyme belonging to the fold-type III group of pyridoxal 5'-phosphate (PLP)-dependent enzymes, has been shown to catalyze the interconversion between L- and D-Alanine. The alanine racemase from the pathogenic bacterium Enterococcus faecalis v583 has been overexpressed in E. coli and was shown to crystallize an enzyme at 295 K, using polyethylene glycol (PEG) 8000 as a precipitant. X-ray diffraction data to 2.5 A has been collected using synchrotron radiation. The crystal is a member of the orthorhombic space group, C222(1), with unit cell parameter of a=94.634, b=156.516, c=147.878 A, and alpha=beta;=gamma=90 degrees. Two or three monomers are likely to be present in the asymmetric unit, with a corresponding Vm of 3.38 A3 Da(-1) and 2.26 A Da(-1) and a solvent content of 63.7% and 45.5%, respectively.

Bioorg Med Chem Lett. 2008 Feb 15; 18(4): 1350-3
Fichna J, do-Rego JC, Janecki T, Staniszewska R, Poels J, Broeck JV, Costentin J, Schiller PW, Janecka A

The mu-opioid agonists endomorphin-1 (Tyr-Pro-Trp-Phe-NH(2)) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)) exhibit an extremely high selectivity for the mu-opioid receptor and thus represent a potential framework for modification into mu-antagonists. Here we report on the synthesis and biological evaluation of novel [d-2-Nal(4)]endomorphin-2 analogs, [Sar(2),d-2-Nal(4)]endomorphin-2 and [Dmt(1),Sar(2),d-2-Nal(4)]endomorphin-2 (Dmt=2'6'-dimethyltyrosine; Sar=N-methylglycine, sarcosine; d-2-Nal=3-(2-naphthyl)-D-Alanine). [Dmt(1),Sar(2),d-2-Nal(4)]endomorphin-2 possessed very high affinity for the mu-opioid receptor (IC(50)=0.01+/-0.001 nM) and turned out to be a potent and extremely selective mu-opioid receptor antagonist, as judged by the in vitro aequorin luminescence-based calcium assay (pA(2)=9.19). However, in the in vivo hot plate test in mice this analog was less potent than our earlier mu-opioid receptor antagonist, [Dmt(1),d-2-Nal(4)]endomorphin-2 (antanal-2). The exceptional mu-opioid receptor in vitro activity and selectivity of [Dmt(1), Sar(2),d-2-Nal(4)]endomorphin-2 makes this analog a valuable pharmacological tool, but further modifications are needed to improve its in vivo profile.

J Org Chem. 2008 Feb 1; 73(3): 1162-4
Hoang CT, Nguyen VH, Alezra V, Kouklovsky C

The antipsychotic compound nemonapride 1 was synthesized in nine steps from D-Alanine 2. The key steps for the synthesis of the 3-aminopyrrolidine moiety include a Birch reduction of a cyclic enaminoester and the reduction of a pyrrolidinone to the pyrrolidine 7. Final coupling with the benzoic acid derivative 9 gave 1 as a single enantio- and diastereomer.

J Mol Biol. 2008 Feb 15; 376(2): 405-13
Macheboeuf P, Lemaire D, Martins Ados S, Dideberg O, Jamin M, Dessen A

Class A penicillin-binding proteins (PBPs) catalyze the last two steps in the biosynthesis of peptidoglycan, a key component of the bacterial cell wall. Both reactions, glycosyl transfer (polymerization of glycan chains) and transpeptidation (cross-linking of stem peptides), are essential for peptidoglycan stability and for the cell division process, but remain poorly understood. The PBP-catalyzed transpeptidation reaction is the target of beta-lactam antibiotics, but their vast employment worldwide has prompted the appearance of highly resistant strains, thus requiring concerted efforts towards an understanding of the transpeptidation reaction with the goal of developing better antibacterials. This goal, however, has been elusive, since PBP substrates are rapidly deacylated. In this work, we provide a structural snapshot of a "trapped" covalent intermediate of the reaction between a class A PBP with a pseudo-substrate, N-benzoyl-D-alanylmercaptoacetic acid thioester, which partly mimics the stem peptides contained within the natural, membrane-associated substrate, lipid II. The structure reveals that the D-alanyl moiety of the covalent intermediate (N-benzoyl-D-Alanine) is stabilized in the cleft by a network of hydrogen bonds that place the carbonyl group in close proximity to the oxyanion hole, thus mimicking the spatial arrangement of beta-lactam antibiotics within the PBP active site. This arrangement allows the target bond to be in optimal position for attack by the acceptor peptide and is similar to the structural disposition of beta-lactam antibiotics with PBP clefts. This information yields a better understanding of PBP catalysis and could provide key insights into the design of novel PBP inhibitors.

Biochemistry. 2008 Jan 22; 47(3): 965-77
Colussi T, Parsonage D, Boles W, Matsuoka T, Mallett TC, Karplus PA, Claiborne A

The FAD-dependent alpha-glycerophosphate oxidase (GlpO) from Enterococcus casseliflavus and Streptococcus sp. was originally studied as a soluble flavoprotein oxidase; surprisingly, the GlpO sequence is 30-43% identical to those of the alpha-glycerophosphate dehydrogenases (GlpDs) from mitochondrial and bacterial sources. The structure of a deletion mutant of Streptococcus sp. GlpO (GlpODelta, lacking a 50-residue insert that includes a flexible surface region) has been determined using multiwavelength anomalous dispersion data and refined at 2.3 A resolution. Using the GlpODelta structure as a search model, we have also determined the intact GlpO structure, as refined at 2.4 A resolution. The first two domains of the GlpO fold are most closely related to those of the flavoprotein glycine oxidase, where they function in FAD binding and substrate binding, respectively; the GlpO C-terminal domain consists of two helix bundles and is not closely related to any known structure. The flexible surface region in intact GlpO corresponds to a segment of missing electron density that links the substrate-binding domain to a betabetaalpha element of the FAD-binding domain. In accordance with earlier biochemical studies (stabilizations of the covalent FAD-N5-sulfite adduct and p-quinonoid form of 8-mercapto-FAD), Ile430-N, Thr431-N, and Thr431-OG are hydrogen bonded to FAD-O2alpha in GlpODelta, stabilizing the negative charge in these two modified flavins and facilitating transfer of a hydride to FAD-N5 (from Glp) as well. Active-site overlays with the glycine oxidase-N-acetylglycine and d-amino acid oxidase-D-Alanine complexes demonstrate that Arg346 of GlpODelta is structurally equivalent to Arg302 and Arg285, respectively; in both cases, these residues interact directly with the amino acid substrate or inhibitor carboxylate. The structural and functional divergence between GlpO and the bacterial and mitochondrial GlpDs is also discussed.

Biotechnol Bioeng. 2008 Jun 1; 100(2): 223-30
Chien LJ, Lee CK

Rapid recovery, immobilization, and silica encapsulation of a dual-fusion enzyme was achieved by using iminodiacetic acid (IDA) modified magnetic nanoparticle as a carrier. D-amino acid oxidase (DAAO) of Rhodosporidium toruloides was used as a model enzyme in which a silica-precipitating peptide R5 and a metal ion complexing peptide (His)(6) were fused to its N- and C-terminal, respectively. After charging the magnetic particle with Cu(2+), the dual-fusion DAAO of 0.43 g could be directly recovered from the recombinant E. coli crude extract and immobilized on 1 g of the magnetic particle. Once in contact with hydrolyzed tetramethoxysilane (TMOS), the homogeneously dispersed immobilized dual-fusion DAAO was biosilicificated to form aggregates with size about 50 microm. The silica-encapsulated immobilized DAAO demonstrated a pyruvic acid production rate comparable with that of the naked immobilized DAAO in five repeated batch reactions when D-Alanine was used as substrate. Furthermore, 85% of its activity remained after incubation at 60 degrees C for 1 h while the naked immobilized DAAO lost all its activity. This process provides the advantages that recombinant fusion enzyme can be directly recovered from crude extract, silica encapsulation protects the enzyme from leakage and denaturation, and the enzyme activity can be easily retrieved by applying a magnetic field.

J Biol Chem. 2008 Mar 7; 283(10): 6402-17
Lloyd AJ, Gilbey AM, Blewett AM, De Pascale G, El Zoeiby A, Levesque RC, Catherwood AC, Tomasz A, Bugg TD, Roper DI, Dowson CG

MurM is an aminoacyl ligase that adds l-serine or l-alanine as the first amino acid of a dipeptide branch to the stem peptide lysine of the pneumococcal peptidoglycan. MurM activity is essential for clinical pneumococcal penicillin resistance. Analysis of peptidoglycan from the highly penicillin-resistant Streptococcus pneumoniae strain 159 revealed that in vivo and in vitro, in the presence of the appropriate acyl-tRNA, MurM(159) alanylated the peptidoglycan epsilon-amino group of the stem peptide lysine in preference to its serylation. However, in contrast, identical analyses of the penicillin-susceptible strain Pn16 revealed that MurM(Pn16) activity supported serylation more than alanylation both in vivo and in vitro. Interestingly, both MurM(Pn16) acylation activities were far lower than the alanylation activity of MurM(159). The resulting differing stem peptide structures of 159 and Pn16 were caused by the profoundly greater catalytic efficiency of MurM(159) compared with MurM(Pn16) bought about by sequence variation between these enzymes and, to a lesser extent, differences in the in vivo tRNA(Ala):tRNA(Ser) ratio in 159 and Pn16. Kinetic analysis revealed that MurM(159) acted during the lipid-linked stages of peptidoglycan synthesis, that the d-alanyl-D-Alanine of the stem peptide and the lipid II N-acetylglucosaminyl group were not essential for substrate recognition, that epsilon-carboxylation of the lysine of the stem peptide was not tolerated, and that lipid II-alanine was a substrate, suggesting an evolutionary link to staphylococcal homologues of MurM such as FemA. Kinetic analysis also revealed that MurM recognized the acceptor stem and/or the TPsiC loop stem of the tRNA(Ala). It is anticipated that definition of the minimal structural features of MurM substrates will allow development of novel resistance inhibitors that will restore the efficacy of beta-lactams for treatment of pneumococcal infection.

J Microbiol Biotechnol. 2007 May; 17(5): 830-9
Kim BC, Lee JM, Ahn JS, Kim BS

Pradimicins are potent antifungal antibiotics having an unusual dihydrobenzo[alpha]naphthacenequinone aglycone substituted with D-Alanine and sugars. Pradimicins are polyketide antibiotics produced by Actinomadura hibisca P157-2. The gene cluster involved in the biosynthesis of pradimicins was cloned and sequenced. The pradimicin gene cluster was localized to a 39-kb DNA segment and its involvement in the biosynthesis of pradimicin was proven by gene inactivation of prmA and prmB (ketosynthases alpha and beta). The pradimicin gene cluster consists of 28 open reading frames (ORFs), encoding a type II polyketide synthase (PKS), the enzymes involved in sugar biosynthesis and tailoring enzymes as well as two resistance proteins. The deduced proteins showed strong similarities to the previously validated gene clusters of angucyclic polyketides such as rubromycin, griseorhodin, and fredericamycin. From the pradimicin gene cluster, prmP3 encoding a component of the acetyl-CoA carboxylase complex was disrupted. The production levels of pradimicins of the resulting mutants decreased to 62% of the level produced by the wild-type strain, which indicate that the acetyl-CoA carboxylase gene would have a significant role in the production of pradimicins through supplying the extender unit precursor, malonyl-CoA.

J Microbiol Biotechnol. 2007 Jun; 17(6): 934-44
Seul KJ, Park SH, Ryu CM, Lee YH, Ghim SY

Paenibacillus polymyxa E681 is known to be able to suppress plant diseases by producing antimicrobial compounds and to promote plant growth by producing phytohormones, and secreting diverse degrading enzymes. In spite of these capabilities, little is known regarding the flow of information from the bacterial strain to the barley roots. In an attempt to determine the flow of information from the bacterial strain to barley roots, the train was grown in the presence and absence of barley, and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and MALDI-TOF mass spectrometry were used. 2D-PAGE detected approximately 1000 spots in the cell and 1100 spots in the supernatant at a pH 4-10 gradient. Interestingly, about 80 spots from each sample showed quantitative variations. Fifty-three spots from these were analyzed by MALDI-TOF mass spectrometry and 28 proteins were identified. Most of the cytosolic proteins expressed at higher levels were found in P. polymyxa E681 cells grown in the presence of barley rather than in the absence of barley. Proteins detected at a lower level in the surpernatant of P. polymyxa E681 cells grown in the presence of barley were lipoprotein, glucose-6-phosphate 1-dehydrogenase, heat-shock protein HtpG spermidine synthase, OrfZ, ribonuclease PH, and coenzyme PQQ synthesis protein, and flagellar hook-associated protein 2 whereas proteins detected at a higher level in the surpernatant of P. polymyxa E681 cells grown in the presence of barley included D-alanyl-D-Alanine ligase A, isopentenyldiphosphate delta-isomerase, ABC transporter ATP-binding protein Uup, lipase. Many of the proteins belonging to plant-induced stimulons are associated with biosynthetic metabolism and metabolites of proteins and transport. Some of these proteins would be expected to be induced by environmental changes resulting from the accumulation of plant-secreted substances.

J Proteome Res. 2007 Dec; 6(12): 4608-14
Halouska S, Chacon O, Fenton RJ, Zinniel DK, Barletta RG, Powers R

D-Cycloserine (DCS) is only used with multidrug-resistant strains of tuberculosis because of serious side effects. DCS is known to inhibit cell wall biosynthesis, but the in vivo lethal target is still unknown. We have applied NMR-based metabolomics combined with principal component analysis to monitor the in vivo effect of DCS on Mycobacterium smegmatis. Our analysis suggests DCS functions by inhibiting multiple protein targets.

Biochem J. 2008 Jan 15; 409(2): 399-406
Ito T, Hemmi H, Kataoka K, Mukai Y, Yoshimura T

YGL196W of Saccharomyces cerevisiae encodes a putative protein that is unidentified but is predicted to have a motif similar to that of the N-terminal domain of the bacterial alanine racemase. In the present study we found that YGL196W encodes a novel D-serine dehydratase, which belongs to a different protein family from that of the known bacterial enzyme. The yeast D-serine dehydratase purified from recombinant Escherichia coli cells depends on pyridoxal 5'-phosphate and zinc, and catalyses the conversion of D-serine into pyruvate and ammonia with the K(m) and k(cat) values of 0.39 mM and 13.1 s(-1) respectively. D-Threonine and beta-Cl-D-Alanine also serve as substrates with catalytic efficiencies which are approx. 3 and 2% of D-serine respectively. L-Serine, L-threonine and beta-Cl-L-alanine are inert as substrates. Atomic absorption analysis revealed that the enzyme contains one zinc atom per enzyme monomer. The enzyme activities toward D-serine and D-threonine were decreased by EDTA treatment and recovered by the addition of Zn2+. Little recovery was observed with Mg2+, Mn2+, Ca2+, Ni2+, Cu2+, K+ or Na+. In contrast, the activity towards beta-Cl-D-Alanine was retained after EDTA treatment. These results suggest that zinc is involved in the elimination of the hydroxy group of D-serine and D-threonine. D-Serine dehydratase of S. cerevisiae is probably the first example of a eukaryotic D-serine dehydratase and that of a specifically zinc-dependent pyridoxal enzyme as well.

Clin Vaccine Immunol. 2007 Dec; 14(12): 1629-33
Deininger S, Figueroa-Perez I, Sigel S, Stadelmaier A, Schmidt RR, Hartung T, von Aulock S

Lipoteichoic acid (LTA) from gram-positive bacteria is the counterpart to lipopolysaccharide from gram-negative bacteria. LTA, which activates Toll-like receptor 2 (TLR2), induces a unique cytokine and chemokine pattern. The chemical synthesis of LTA proved its immunostimulatory properties. To determine the minimal active structure of LTA, we reduced synthetic LTA in a number of steps down to the synthetic anchor and employed these molecules to stimulate interleukin-8 (IL-8) release in human whole blood. Ten times more of the synthetic structures with four to six D-Alanine-substituted polyglycerophosphate units (50 nM) than of the native LTA preparation was required to induce IL-8 release. A further reduction to three backbone units with two or no D-Alanine residues resulted in cytokine induction only from 500 nM. The synthetic anchor was not able to induce IL-8 release even at 5 muM. When the LTA derivatives were used at 500 nM, they induced increasing levels of IL-8 and tumor necrosis factor alpha with increasing elongation of the backbone. Peritoneal macrophages were less responsive than human blood to the synthetic structures. Therefore, TLR2 dependency could be shown only with cells from TLR2-deficient mice for the two largest synthetic structures. This was confirmed by using TLR2-transfected HEK 293 cells. Taken together, these data indicate that although the synthetic anchor (which, unlike the native anchor, contains only myristic acid) cannot induce cytokine release, the addition of three backbone units, even without D-Alanine substituents, confers this ability. Lengthening of the chain with D-Alanine-substituted backbone units results in increased cytokine-inducing potency and a more sensitive response.

Infect Immun. 2007 Dec; 75(12): 5726-34
McKevitt MT, Bryant KM, Shakir SM, Larabee JL, Blanke SR, Lovchik J, Lyons CR, Ballard JD

Bacillus anthracis transitions from a dormant spore to a vegetative bacillus through a series of structural and biochemical changes collectively referred to as germination. The timing of germination is important during early steps in infection and may determine if B. anthracis survives or succumbs to responsive macrophages. In the current study experiments determined the contribution of endogenous D-Alanine production to the efficiency and timing of B. anthracis spore germination under in vitro and in vivo conditions. Racemase-mediated production of endogenous D-Alanine by B. anthracis altered the kinetics for initiation of germination over a range of spore densities and exhibited a threshold effect wherein small changes in spore number resulted in major changes in germination efficiency. This threshold effect correlated with D-Alanine production, was prevented by an alanine racemase inhibitor, and required L-alanine. Interestingly, endogenous production of inhibitory levels of D-Alanine was detected under experimental conditions that did not support germination and in a germination-deficient mutant of B. anthracis. Racemase-dependent production of D-Alanine enhanced survival of B. anthracis during interaction with murine macrophages, suggesting a role for inhibition of germination during interaction with these cells. Finally, in vivo experiments revealed an approximately twofold decrease in the 50% lethal dose of B. anthracis spores administered in the presence of D-Alanine, indicating that rates of germination may be directly influenced by the levels of this amino acid during early stages of disease.

Chemistry. 2007; 13(35): 9791-823
Tamiya M, Ohmori K, Kitamura M, Kato H, Arai T, Oorui M, Suzuki K

A general approach to the regio- and stereoselective total synthesis of the benanomicin-pradimicin antibiotics (BpAs) is described. Construction of the aglycon has been achieved by 1) the diastereoselective ring-opening of a biaryl lactone by using (R)-valinol as a chiral nucleophile and 2) the stereocontrolled semi-pinacol cyclization of the aldehyde acetal by using SmI(2) in the presence of BF(3)OEt(2) and a proton source to afford the ABCD tetracyclic monoprotected diol. This strategy enabled us to control the two stereogenic sites in the B ring (C-5 and C-6) and the regioselective introduction of the carbohydrate moiety. The ABCD tetracycle could serve as an ideal platform for the divergent access to various BpAs. The amino acid (D-Alanine) was introduced onto the ABCD tetracycle. Glycosylation was promoted by the combination of Cp(2)HfCl(2) and AgOTf (1:2 ratio). Construction of the E ring followed by deprotection completed the first total synthesis of benanomicin A (2 a), benanomicin B (2 b), and pradimicin A (1 a). The route is flexible enough to allow the synthesis of other congeners differing in their amino acid and carbohydrate moieties.

ACS Chem Biol. 2007 Sep 21; 2(9): 620-4
Kumar I, Josephine HR, Pratt RF

The membrane-bound bacterial D-alanyl- D-Alanine peptidases or penicillin-binding proteins (PBPs) catalyze the final transpeptidation reaction of bacterial cell wall biosynthesis and are the targets of beta-lactam antibiotics. Rather surprisingly, the substrate specificity of these enzymes is not well understood. In this paper, we present measurements of the reactivity of typical examples of these enzymes with peptidoglycan-mimetic beta-lactams under in vivo conditions. The minimum inhibitory concentrations of beta-lactams with Escherichia coli-specific side chains were determined against E. coli cells. Analogous measurements were made with Streptococcus pneumoniae R6. The reactivity of the relevant beta-lactams with E. coli PBPs in membrane preparations was also determined. The results show that under none of the above protocols were beta-lactams with peptidoglycan-mimetic side chains more reactive than generic analogues. This suggests that in vivo, as in vitro, these enzymes do not specifically recognize elements of peptidoglycan structure local to the reaction center. Substrate recognition must thus involve extended structure.

Appl Microbiol Biotechnol. 2007 Nov; 77(2): 355-66
Zhang X, Jantama K, Moore JC, Shanmugam KT, Ingram LO

Escherichia coli W was genetically engineered to produce L: -alanine as the primary fermentation product from sugars by replacing the native D: -lactate dehydrogenase of E. coli SZ194 with alanine dehydrogenase from Geobacillus stearothermophilus. As a result, the heterologous alanine dehydrogenase gene was integrated under the regulation of the native D: -lactate dehydrogenase (ldhA) promoter. This homologous promoter is growth-regulated and provides high levels of expression during anaerobic fermentation. Strain XZ111 accumulated alanine as the primary product during glucose fermentation. The methylglyoxal synthase gene (mgsA) was deleted to eliminate low levels of lactate and improve growth, and the catabolic alanine racemase gene (dadX) was deleted to minimize conversion of L: -alanine to D: -alanine. In these strains, reduced nicotinamide adenine dinucleotide oxidation during alanine biosynthesis is obligately linked to adenosine triphosphate production and cell growth. This linkage provided a basis for metabolic evolution where selection for improvements in growth coselected for increased glycolytic flux and alanine production. The resulting strain, XZ132, produced 1,279 mmol alanine from 120 g l(-1) glucose within 48 h during batch fermentation in the mineral salts medium. The alanine yield was 95% on a weight basis (g g(-1) glucose) with a chiral purity greater than 99.5% L: -alanine.