DdlN from Vancomycin-Producing Amycolatopsis orientalis C329.2 Is a VanA Homologue with d-Alanyl-d-Lactate Ligase Activity

Vancomycin-resistant enterococci acquire high-level resistance to glycopeptide antibiotics through the synthesis of peptidoglycan terminating in d-alanyl-d-lactate. A key enzyme in this process is a d-alanyl-d-alanine ligase homologue, VanA or VanB, which preferentially catalyzes the synthesis of the depsipeptide d-alanyl-d-lactate. We report the overexpression, purification, and enzymatic characterization of DdlN, a VanA and VanB homologue encoded by a gene of the vancomycin-producing organism Amycolatopsis orientalis C329.2. Evaluation of kinetic parameters for the synthesis of peptides and depsipeptides revealed a close relationship between VanA and DdlN in that depsipeptide formation was kinetically preferred at physiologic pH; however, the DdlN enzyme demonstrated a narrower substrate specificity and commensurately increased affinity for d-lactate in the C-terminal position over VanA. The results of these functional experiments also reinforce the results of previous studies that demonstrated that glycopeptide resistance enzymes from glycopeptide-producing bacteria are potential sources of resistance enzymes in clinically relevant bacteria.The origin of antibiotic resistance determinants is of significant interest for several reasons, including the prediction of the emergence and spread of resistance patterns, the design of new antimicrobial agents, and the identification of potential reservoirs for resistance elements. Antibiotic resistance can occur either through spontaneous mutation in the target or by the acquisition of external genetic elements such as plasmids or transposons which carry resistance genes (7). The origins of these acquired genes are varied, but it has long been recognized that potential reservoirs are antibiotic-producing organisms which naturally harbor antibiotic resistance genes to protect themselves from the actions of toxic compounds (6).High-level resistance to glycopeptide antibiotics such as vancomycin and teicoplanin in vancomycin-resistant enterococci (VRE) is conferred by the presence of three genes, vanH, vanA (or vanB), and vanX, which, along with auxiliary genes necessary for inducible gene expression, are found on transposons integrated into plasmids or the bacterial genome (1, 20). These three genes are essential to resistance and serve to change the C-terminal peptide portion of the peptidoglycan layer from d-alanyl-d-alanine (d-Ala-d-Ala) to d-alanyl-d-lactate (d-Ala-d-Lac). This change results in the loss of a critical hydrogen bond between vancomycin and the d-Ala-d-Ala terminus and in a 1,000-fold decrease in binding affinity between the antibiotic and the peptidoglycan layer, which is the basis for the bactericidal action of this class of compounds (5). The vanH gene encodes a d-lactate dehydrogenase which provides the requisite d-Lac (3, 5), while the vanX gene encodes a highly specific dd-peptidase which cleaves only d-Ala-d-Ala produced endogenously while leaving d-Ala-d-Lac intact (19, 21). The final gene, vanA or vanB, encodes an ATP-dependent d-Ala-d-Lac ligase (4, 8, 10). This enzyme has sequence homology with the chromosomal d-Ala-d-Ala ligases, which are essential for peptidoglycan synthesis but which generally lack the ability to synthesize d-Ala-d-Lac (9).We have recently cloned vanH, vanA, and vanX homologues from two glycopeptide antibiotic-synthesizing organisms: Amycolatopsis orientalis C329.2, which produces vancomycin, and Streptomyces toyocaensis NRRL 15009, which produces {"type":"entrez-nucleotide","attrs":{"text":"A47934","term_id":"2301796","term_text":"A47934"}}A47934 (14). In addition, the vanH-vanA-vanX gene cluster was identified in several other glycopeptide producers. We have also demonstrated that the VanA homologue from S. toyocaensis NRRL 15009 can synthesize d-Ala-d-Lac in vitro and in the glycopeptide-sensitive host Streptomyces lividans (15, 16). We now report the expression of the A. orientalis C329.2 VanA homologue DdlN in Escherichia coli, its purification, and its enzymatic characterization. These data reinforce the striking similarity between vancomycin resistance elements in VRE and glycopeptide-producing organisms and support the possibility of a common origin for these enzymes.

Expression, purification, and specificity of DdlN.

DdlN was overexpressed in E. coli under the control of the bacteriophage T7 promoter. The construct gave good yields of highly purified enzyme following a four-step purification procedure (Table ​(Table1;1; Fig. ​Fig.1).1). Like other dd-ligases, DdlN behaved like a dimer in solution (not shown).

TABLE 1

Purification of DdlN from E. coli BL21 (DE3)/pETDdlN

Molecular Basis of Vancomycin Dependence in VanA-Type Staphylococcus aureus VRSA-9

The vancomycin-resistant Staphylococcus aureus VRSA-9 clinical isolate was partially dependent on glycopeptide for growth. The responsible vanA operon had the same organization as that of Tn1546 and was located on a plasmid. The chromosomal d-Ala:d-Ala ligase (ddl) gene had two point mutations that led to Q260K and A283E substitutions, resulting in a 200-fold decrease in enzymatic activity compared to that of the wild-type strain VRSA-6. To gain insight into the mechanism of enzyme impairment, we determined the crystal structure of VRSA-9 Ddl and showed that the A283E mutation induces new ion pair/hydrogen bond interactions, leading to an asymmetric rearrangement of side chains in the dimer interface. The Q260K substitution is located in an exposed external loop and did not induce any significant conformational change. The VRSA-9 strain was susceptible to oxacillin due to synthesis of pentadepsipeptide precursors ending in d-alanyl-d-lactate which are not substrates for the β-lactam-resistant penicillin binding protein PBP2′. Comparison with the partially vancomycin-dependent VRSA-7, whose Ddl is 5-fold less efficient than that of VRSA-9, indicated that the levels of vancomycin dependence and susceptibility to β-lactams correlate with the degree of Ddl impairment. Ddl drug targeting could therefore be an effective strategy against vancomycin-resistant S. aureus.Methicillin-resistant Staphylococcus aureus (MRSA) bacteria that have acquired the vancomycin resistance vanA operon from glycopeptide-resistant enterococci are designated vancomycin-resistant S. aureus (VRSA) (29). Vancomycin acts by binding to the C-terminal acyl-d-Ala-d-Ala of the undecaprenol-diphosphate MurNAc-pentapeptide intermediate and inhibits transglycosylation and transpeptidation reactions in cell wall peptidoglycan polymerization and cross-linking (30). d-Ala-d-Ala is synthesized by the ATP-dependent d-Ala:d-Ala ligase (Ddl) (EC 6.3.2.4) before its incorporation in peptidoglycan precursors (26, 35). VanA-type vancomycin resistance results from the incorporation into peptidoglycan intermediates of a d-alanyl-d-lactate (d-Ala-d-Lac) depsipeptide, synthesized by a d-Ala:d-Lac ligase, which is responsible for diminished binding affinity of glycopeptides for their target. Kinetic analyses of Ddls have established two subsites in the active site for d-Ala binding (24, 27). The reaction mechanism culminates in the transfer of the γ-phosphoryl of ATP to the carboxyl group of d-Ala₁ to produce an acylphosphate and ADP. The acyl carbon atom of the acylphosphate then reacts with the amino group of d-Ala₂ to yield a tetrahedral intermediate. Finally, the intermediate releases phosphate to yield d-Ala-d-Ala.Mutants of Enterococcus faecium (8, 14), Enterococcus faecalis (34), and S. aureus (23) with an impaired Ddl are able to grow because they use the vancomycin resistance pathway for cell wall synthesis. Since resistance is inducible by the drug, these bacteria require the presence of vancomycin in the culture medium for growth. Ddls from vancomycin-dependent enterococci (14) have mutations affecting amino acids highly conserved in the d-Ala:d-Ala ligase superfamily (10). Molecular modeling based on the X-ray structure of Escherichia coli DdlB (11) revealed that all the mutated residues interact directly with one of the substrates of the enzymatic reaction or stabilize the position of critical residues in the active site. However, the degree of enzyme impairment was not evaluated biochemically. Recently, we reported the mechanism of vancomycin dependence in VanA-type S. aureus VRSA-7 and showed that the chromosomal Ddl had the single mutation N308K, which probably affects the binding of the transition-state intermediate, leading to a 1,000-fold decrease in activity relative to that of the wild-type enzyme (23). Glycopeptide-dependent mutants could therefore be considered useful tools to explore structure-activity relationships of the Ddl, which represents an attractive target for designing new drugs. Here we describe the partially vancomycin-dependent VanA-type S. aureus strain VRSA-9 and report the biochemical and structural characterization of its mutated Ddl.     

Crystal structure of a zinc-dependent D-serine dehydratase from chicken kidney

d-Serine is a physiological co-agonist of the N-methyl-d-aspartate receptor. It regulates excitatory neurotransmission, which is important for higher brain functions in vertebrates. In mammalian brains, d-amino acid oxidase degrades d-serine. However, we have found recently that in chicken brains the oxidase is not expressed and instead a d-serine dehydratase degrades d-serine. The primary structure of the enzyme shows significant similarities to those of metal-activated d-threonine aldolases, which are fold-type III pyridoxal 5′-phosphate (PLP)-dependent enzymes, suggesting that it is a novel class of d-serine dehydratase. In the present study, we characterized the chicken enzyme biochemically and also by x-ray crystallography. The enzyme activity on d-serine decreased 20-fold by EDTA treatment and recovered nearly completely by the addition of Zn²⁺. None of the reaction products that would be expected from side reactions of the PLP-d-serine Schiff base were detected during the >6000 catalytic cycles of dehydration, indicating high reaction specificity. We have determined the first crystal structure of the d-serine dehydratase at 1.9 Å resolution. In the active site pocket, a zinc ion that coordinates His³⁴⁷ and Cys³⁴⁹ is located near the PLP-Lys⁴⁵ Schiff base. A theoretical model of the enzyme-d-serine complex suggested that the hydroxyl group of d-serine directly coordinates the zinc ion, and that the ϵ-NH₂ group of Lys⁴⁵ is a short distance from the substrate Cα atom. The α-proton abstraction from d-serine by Lys⁴⁵ and the elimination of the hydroxyl group seem to occur with the assistance of the zinc ion, resulting in the strict reaction specificity.     

vanI: a novel d-Ala-d-Lac vancomycin resistance gene cluster found in Desulfitobacterium hafniense

The glycopeptide vancomycin was until recently considered a drug of last resort against Gram-positive bacteria. Increasing numbers of bacteria, however, are found to carry genes that confer resistance to this antibiotic. So far, 10 different vancomycin resistance clusters have been described. A chromosomal vancomycin resistance gene cluster was previously described for the anaerobic Desulfitobacterium hafniense Y51. We demonstrate that this gene cluster, characterized by its d-Ala-d-Lac ligase-encoding vanI gene, is present in all strains of D. hafniense, D. chlororespirans and some strains of Desulfosporosinus spp. This gene cluster was not found in vancomycin-sensitive Desulfitobacterium or Desulfosporosinus spp., and we show that this antibiotic resistance can be exploited as an intrinsic selection marker for Desulfitobacterium hafniense and D. chlororespirans. The gene cluster containing vanI is phylogenetically only distantly related with those described from soil and gut bacteria, but clusters instead with vancomycin resistance genes found within the phylum Actinobacteria that include several vancomycin-producing bacteria. It lacks a vanH homologue, encoding a D-lactate dehydrogenase, previously thought to always be present within vancomycin resistance gene clusters. The location of vanH outside the resistance gene cluster likely hinders horizontal gene transfer. Hence, the vancomycin resistance cluster in D. hafniense should be regarded a novel one that we here designated vanI after its unique d-Ala-d-Lac ligase.     

Biochemical and Biophysical Characterization of an Unexpected Bacteriolytic Activity of VanX,a Member of the Vancomycin-resistance vanA Gene Cluster

VanX is a d-alanyl-d-alanine (d-Ala–d-Ala) dipeptidase encoded in the vancomycin-resistance vanA gene cluster. Here we report that strong bacteriolysis occurred when isolated VanX was expressed in Escherichia coli at temperatures lower than 30 °C, which was unexpected because the vanA operon confers vancomycin resistance by protecting the cell wall. Therefore, we monitored cell lysis by measuring sample turbidity with absorbance at 590 nm and VanX expression using SDS-PAGE. No cell lysis was observed when VanX was expressed, even in large quantities, in the cell inclusion bodies at 37 °C, suggesting that a natively folded VanX is required for lysis. In addition, VanX mutants with suppressed dipeptidase activity did not lyse E. coli cells, confirming that bacteriolysis originated from the dipeptidase activity of VanX. We also observed shape changes in E. coli cells undergoing VanX-mediated lysis with optical microscopy and classified these changes into three classes: bursting, deformation, and leaking fluid. Optical microscopic image analysis fully corroborated our interpretation of the turbidity changes in the samples. From a practical perspective, the finding that VanX expressed in isolation induces cell lysis suggests that inhibitors of VanA and VanH that act downstream from VanX could provide a new class of therapeutic chemicals against bacteria expressing the vancomycin-resistance gene cluster.     

Specificity Determinants for Lysine Incorporation in Staphylococcus aureus Peptidoglycan as Revealed by the Structure of a MurE Enzyme Ternary Complex

Formation of the peptidoglycan stem pentapeptide requires the insertion of both l and d amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between l-lysine and d,l-diaminopimelic acid, the predominant amino acid that replaces l-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of l-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for l-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic l-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.     

A Novel Type of Peptidoglycan-binding Domain Highly Specific for Amidated d-Asp Cross-bridge,Identified in Lactobacillus casei Bacteriophage Endolysins

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-l-alanine amidase, whereas Lc-Lys-2 is a γ-d-glutamyl-l-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with d-Ala⁴→d-Asx-l-Lys³ in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting d-Ala⁴→l-Ala-(l-Ala/l-Ser)-l-Lys³; moreover, they do not lyse the L. lactis mutant containing only the nonamidated d-Asp cross-bridge, i.e. d-Ala⁴→d-Asp-l-Lys³. In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 l-Lys³-d-Asn-l-Lys³ bridges replacing the wild-type 4→3 d-Ala⁴-d-Asn-l-Lys³ bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly d-Asn but not PG with only the nonamidated d-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the d-Asn interpeptide bridge of PG.     

Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K_m for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm.     

Autolysis of cell walls of Bacillus stearothermophilus B65 and the chemical structure of the peptidoglycan

1. The cell walls of Bacillus stearothermophilus B65 contain glucosamine, muramic acid, alanine, α-diaminopimelic acid (Dap), glutamic acid, aspartic acid, glycine, and serine in the molecular proportions 0.60:0.64:2.30:0.85:1.00:0.11:0.13:0.31. 2. Both d- and l-alanine are present, but glutamic acid and diaminopimelic acid are present only as the d- and meso-isomers respectively. 3. The peptide fragments Ala-Dap, Dap-Ala, and Dap-Ala-Dap have been isolated from a partial acid hydrolysate of the cell walls. 4. The major products of autolysis of the cell wall were d-alanine, a peptide mixture, peptidoglycan material and a peptidoglycan–teichoic acid complex. 5. Separation of the peptide mixture into ten major peptides was achieved by DEAE-Sephadex and paper chromatography, and paper electrophoresis. 6. The structures of these peptides have been determined and they fall into four groups, the individual members of each group differing only in number or position of carboxamide substituents. 7. The structures are I, a tripeptide l-Ala–d-Glu-meso-Dap; II, a pentapeptide made up by the tripeptide (I) linked through the -amino group of its diaminopimelic acid residue to the carboxyterminal of the dipeptide meso-Dap-d-Ala; III, a heptapeptide made up by a similar linkage between the tripeptide (I) and the tetrapeptide l-Ala-d-Glu-meso-Dap-d-Ala; IV, a possible undecapeptide made up by a further tetrapeptide similarly linked to the heptapeptide (III) structure. 8. The structure of the peptidoglycan and the actions of the autolytic enzymes are discussed in terms of these peptide structures.     

The Active Form of E6-associated protein (E6AP)/UBE3A Ubiquitin Ligase Is an Oligomer

Employing ¹²⁵I-polyubiquitin chain formation as a functional readout of ligase activity, biochemical and biophysical evidence demonstrates that catalytically active E6-associated protein (E6AP)/UBE3A is an oligomer. Based on an extant structure previously discounted as an artifact of crystal packing forces, we propose that the fully active form of E6AP is a trimer, analysis of which reveals a buried surface of 7508 Ų and radially symmetric interacting residues that are conserved within the Hect (homologous to E6AP C terminus) ligase superfamily. An absolutely conserved interaction between Phe⁷²⁷ and a hydrophobic pocket present on the adjacent subunit is critical for trimer stabilization because mutation disrupts the oligomer and decreases k_cat 62-fold but fails to affect E2∼ubiquitin binding or subsequent formation of the Hect domain Cys⁸²⁰∼ubiquitin thioester catalytic intermediate. Exogenous N-acetylphenylalanylamide reversibly antagonizes Phe⁷²⁷-dependent trimer formation and catalytic activity (K_i = 12 mm), as does a conserved α-helical peptide corresponding to residues 474–490 of E6AP isoform 1 (K_i = 22 μm) reported to bind the hydrophobic pocket of other Hect ligases, presumably blocking Phe⁷²⁷ intercalation and trimer formation. Conversely, oncogenic human papillomavirus-16/18 E6 protein significantly enhances E6AP catalytic activity by promoting trimer formation (K_activation = 1.5 nm) through the ability of E6 to form homodimers. Recombinant E6 protein additionally rescues the k_cat defect of the Phe⁷²⁷ mutation and that of a specific loss-of-function Angelman syndrome mutation that promotes trimer destabilization. The present findings codify otherwise disparate observations regarding the mechanism of E6AP and related Hect ligases in addition to suggesting therapeutic approaches for modulating ligase activity.     

The Elucidation of the Structure of Thermotoga maritima Peptidoglycan Reveals Two Novel Types of Cross-link

Thermotoga maritima is a Gram-negative, hyperthermophilic bacterium whose peptidoglycan contains comparable amounts of l- and d-lysine. We have determined the fine structure of this cell-wall polymer. The muropeptides resulting from the digestion of peptidoglycan by mutanolysin were separated by high-performance liquid chromatography and identified by amino acid analysis after acid hydrolysis, dinitrophenylation, enzymatic determination of the configuration of the chiral amino acids, and mass spectrometry. The high-performance liquid chromatography profile contained four main peaks, two monomers, and two dimers, plus a few minor peaks corresponding to anhydro forms. The first monomer was the d-lysine-containing disaccharide-tripeptide in which the d-Glu-d-Lys bond had the unusual γ→ϵ arrangement (GlcNAc-MurNAc-l-Ala-γ-d-Glu-ϵ-d-Lys). The second monomer was the conventional disaccharide-tetrapeptide (GlcNAc-MurNAc-l-Ala-γ-d-Glu-l-Lys-d-Ala). The first dimer contained a disaccharide-l-Ala as the acyl donor cross-linked to the α-amine of d-Lys in a tripeptide acceptor stem with the sequence of the first monomer. In the second dimer, donor and acceptor stems with the sequences of the second and first monomers, respectively, were connected by a d-Ala⁴-α-d-Lys³ cross-link. The cross-linking index was 10 with an average chain length of 30 disaccharide units. The structure of the peptidoglycan of T. maritima revealed for the first time the key role of d-Lys in peptidoglycan synthesis, both as a surrogate of l-Lys or meso-diaminopimelic acid at the third position of peptide stems and in the formation of novel cross-links of the l-Ala¹(α→α)d-Lys³ and d-Ala⁴(α→α)d-Lys³ types.Peptidoglycan (or murein) is a giant macromolecule whose main function is the protection of the cytoplasmic membrane against the internal osmotic pressure. It is composed of alternating residues of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc)² cross-linked by short peptides (1). The composition of the peptide stem in nascent peptidoglycan is l-Ala¹-γ-d-Glu²-X³-d-Ala⁴-d-Ala⁵, where X is most often meso-diaminopimelic acid (meso-A₂pm) or l-lysine in Gram-negative and Gram-positive species, respectively (2, 3). In the mature macromolecule, the last d-Ala residue is removed. Cross-linking of the glycan chains generally occurs between the carboxyl group of d-Ala at position 4 of a donor peptide stem and the side-chain amino group of the diamino acid at position 3 of an acceptor peptide stem (4→3 cross-links). Cross-linking is either direct or through a short peptide bridge such as pentaglycine in Staphylococcus aureus (2, 3). The enzymes for the formation of the 4→3 cross-links are active-site serine dd- transpeptidases that belong to the penicillin-binding protein (PBP) family and are the essential targets of β-lactam antibiotics in pathogenic bacteria (4). Catalysis involves the cleavage of the d-Ala⁴-d-Ala⁵ bond of a donor peptide stem and the formation of an amide bond between the carboxyl of d-Ala⁴ and the side chain amine at the third position of an acceptor stem. Transpeptidases of the ld specificity are active-site cysteine enzymes that were shown to act as surrogates of the PBPs in mutants of Enterococcus faecium resistant to β-lactam antibiotics (5). They cleave the X³-d-Ala⁴ bond of a donor stem peptide to form 3→3 cross-links. This alternate mode of cross-linking is usually marginal, although it has recently been shown to predominate in non-replicative “dormant” forms of Mycobacterium tuberculosis (6).Thermotoga maritima is a Gram-negative, extremely thermophilic bacterium isolated from geothermally heated sea floors by Huber et al. (7). A morphological characteristic is the presence of an outer sheath-like envelope called “toga.” Although the organism has received considerable attention for its biotechnological potential, studies about its peptidoglycan are scarce (8–11), and in particular the fine structure of the macromolecule is still unknown. In their initial work, Huber et al. (7) showed that the composition of its peptidoglycan was unusual for a Gram-negative species, because it contained both isomers of lysine and no A₂pm. Recently, we purified and studied the properties of T. maritima MurE (12); this enzyme is responsible for the addition of the amino acid residue at position 3 of the peptide stem (13, 14). We demonstrated that T. maritima MurE added in vitro l- and d-Lys to UDP-MurNAc-l-Ala-d-Glu. Although l-Lys was added in the usual way, yielding the conventional nucleotide UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys containing a d-Glu(γ→α)l-Lys amide bond, the d-isomer was added in an “upside-down” manner, yielding the novel nucleotide UDP-MurNAc-l-Ala-d-Glu(γ→ϵ)d-Lys. We also showed that the d-Lys-containing nucleotide was not a substrate for T. maritima MurF, the subsequent enzyme in the biosynthetic pathway, whereas this ligase catalyzed the addition of dipeptide d-Ala-d-Ala to the l-Lys-containing tripeptide, yielding the conventional UDP-MurNAc-pentapeptide (12).However, both the l-Lys-containing UDP-MurNAc-pentapeptide and d-Lys-containing UDP-MurNAc-tripeptide were used as substrates by T. maritima MraY with comparable efficiencies in vitro (12). This observation implies that the unusual d-Lys-containing peptide stems are likely to be translocated to the periplasmic face of the cytoplasmic membrane and to participate in peptidoglycan polymerization. Therefore, we have determined here the fine structure of T. maritima peptidoglycan and we have shown that l-Lys- and d-Lys-containing peptide stems are both present in the polymer, the latter being involved in the formation of two novel types of peptidoglycan cross-link.     

Utilization of d-Ribitol by Lactobacillus casei BL23 Requires a Mannose-Type Phosphotransferase System and Three Catabolic Enzymes

Lactobacillus casei strains 64H and BL23, but not ATCC 334, are able to ferment d-ribitol (also called d-adonitol). However, a BL23-derived ptsI mutant lacking enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was not able to utilize this pentitol, suggesting that strain BL23 transports and phosphorylates d-ribitol via a PTS. We identified an 11-kb region in the genome sequence of L. casei strain BL23 (LCABL_29160 to LCABL_29270) which is absent from strain ATCC 334 and which contains the genes for a GlpR/IolR-like repressor, the four components of a mannose-type PTS, and six metabolic enzymes potentially involved in d-ribitol metabolism. Deletion of the gene encoding the EIIB component of the presumed ribitol PTS indeed prevented d-ribitol fermentation. In addition, we overexpressed the six catabolic genes, purified the encoded enzymes, and determined the activities of four of them. They encode a d-ribitol-5-phosphate (d-ribitol-5-P) 2-dehydrogenase, a d-ribulose-5-P 3-epimerase, a d-ribose-5-P isomerase, and a d-xylulose-5-P phosphoketolase. In the first catabolic step, the protein d-ribitol-5-P 2-dehydrogenase uses NAD⁺ to oxidize d-ribitol-5-P formed during PTS-catalyzed transport to d-ribulose-5-P, which, in turn, is converted to d-xylulose-5-P by the enzyme d-ribulose-5-P 3-epimerase. Finally, the resulting d-xylulose-5-P is split by d-xylulose-5-P phosphoketolase in an inorganic phosphate-requiring reaction into acetylphosphate and the glycolytic intermediate d-glyceraldehyde-3-P. The three remaining enzymes, one of which was identified as d-ribose-5-P-isomerase, probably catalyze an alternative ribitol degradation pathway, which might be functional in L. casei strain 64H but not in BL23, because one of the BL23 genes carries a frameshift mutation.     

Escherichia coli d-Malate Dehydrogenase,a Generalist Enzyme Active in the Leucine Biosynthesis Pathway

The enzymes of the β-decarboxylating dehydrogenase superfamily catalyze the oxidative decarboxylation of d-malate-based substrates with various specificities. Here, we show that, in addition to its natural function affording bacterial growth on d-malate as a carbon source, the d-malate dehydrogenase of Escherichia coli (EcDmlA) naturally expressed from its chromosomal gene is capable of complementing leucine auxotrophy in a leuB⁻ strain lacking the paralogous isopropylmalate dehydrogenase enzyme. To our knowledge, this is the first example of an enzyme that contributes with a physiologically relevant level of activity to two distinct pathways of the core metabolism while expressed from its chromosomal locus. EcDmlA features relatively high catalytic activity on at least three different substrates (l(+)-tartrate, d-malate, and 3-isopropylmalate). Because of these properties both in vivo and in vitro, EcDmlA may be defined as a generalist enzyme. Phylogenetic analysis highlights an ancient origin of DmlA, indicating that the enzyme has maintained its generalist character throughout evolution. We discuss the implication of these findings for protein evolution.     

Comparison of Predicted Epimerases and Reductases of the Campylobacter jejuni d-altro- and l-gluco-Heptose Synthesis Pathways

Uniquely modified heptoses found in surface carbohydrates of bacterial pathogens are potential therapeutic targets against such pathogens. Our recent biochemical characterization of the GDP-6-deoxy-d-manno- and GDP-6-deoxy-d-altro-heptose biosynthesis pathways has provided the foundation for elucidation of the more complex l-gluco-heptose synthesis pathway of Campylobacter jejuni strain NCTC 11168. In this work we use GDP-4-keto,6-deoxy-d-lyxo-heptose as a surrogate substrate to characterize three enzymes predicted to be involved in this pathway: WcaG_NCTC (also known as Cj1427), MlghB (Cj1430), and MlghC (Cj1428). We compare them with homologues involved in d-altro-heptose production: WcaG₈₁₁₇₆ (formerly WcaG), DdahB (Cjj1430), and DdahC (Cjj1427). We show that despite high levels of similarity, the enzymes have pathway-specific catalytic activities and substrate specificities. MlghB forms three products via C3 and C5 epimerization activities, whereas its DdahB homologue only had C3 epimerase activity along its cognate pathway. MlghC is specific for the double C3/C5 epimer generated by MlghB and produces l-gluco-heptose via stereospecific C4 reductase activity. In contrast, its homologue DdahC only uses the C3 epimer to yield d-altro-heptose via C4 reduction. Finally, we show that WcaG_NCTC is not necessary for l-gluco-heptose synthesis and does not affect its production by MlghB and MlghC, in contrast to its homologue WcaG₈₁₁₇₆, that has regulatory activity on d-altro-heptose synthesis. These studies expand our fundamental understanding of heptose modification, provide new glycobiology tools to synthesize novel heptose derivatives with biomedical applications, and provide a foundation for the structure function analysis of these enzymes.     

Engineering an ATP-dependent <Emphasis Type="SmallCaps">d</Emphasis>-Ala:<Emphasis Type="SmallCaps">d</Emphasis>-Ala ligase for synthesizing amino acid amides from amino acids

We successfully engineered a new enzyme that catalyzes the formation of d-Ala amide (d-AlaNH₂) from d-Ala by modifying ATP-dependent d-Ala:d-Ala ligase (EC 6.3.2.4) from Thermus thermophilus, which catalyzes the formation of d-Ala-d-Ala from two molecules of d-Ala. The new enzyme was created by the replacement of the Ser293 residue with acidic amino acids, as it was speculated to bind to the second d-Ala of d-Ala-d-Ala. In addition, a replacement of the position with Glu performed better than that with Asp with regards to specificity for d-AlaNH₂ production. The S293E variant, which was selected as the best enzyme for d-AlaNH₂ production, exhibited an optimal activity at pH 9.0 and 40 °C for d-AlaNH₂ production. The apparent K _m values of this variant for d-Ala and NH₃ were 7.35 mM and 1.58 M, respectively. The S293E variant could catalyze the synthesis of 9.3 and 35.7 mM of d-AlaNH₂ from 10 and 50 mM d-Ala and 3 M NH₄Cl with conversion yields of 93 and 71.4 %, respectively. This is the first report showing the enzymatic formation of amino acid amides from amino acids.     

Identification and Characterization of d-Hydroxyproline Dehydrogenase and Δ1-Pyrroline-4-hydroxy-2-carboxylate Deaminase Involved in Novel l-Hydroxyproline Metabolism of Bacteria: METABOLIC CONVERGENT EVOLUTION*

l-Hydroxyproline (4-hydroxyproline) mainly exists in collagen, and most bacteria cannot metabolize this hydroxyamino acid. Pseudomonas putida and Pseudomonas aeruginosa convert l-hydroxyproline to α-ketoglutarate via four hypothetical enzymatic steps different from known mammalian pathways, but the molecular background is rather unclear. Here, we identified and characterized for the first time two novel enzymes, d-hydroxyproline dehydrogenase and Δ¹-pyrroline-4-hydroxy-2-carboxylate (Pyr4H2C) deaminase, involved in this hypothetical pathway. These genes were clustered together with genes encoding other catalytic enzymes on the bacterial genomes. d-Hydroxyproline dehydrogenases from P. putida and P. aeruginosa were completely different from known bacterial proline dehydrogenases and showed similar high specificity for substrate (d-hydroxyproline) and some artificial electron acceptor(s). On the other hand, the former is a homomeric enzyme only containing FAD as a prosthetic group, whereas the latter is a novel heterododecameric structure consisting of three different subunits (α₄β₄γ₄), and two FADs, FMN, and [2Fe-2S] iron-sulfur cluster were contained in αβγ of the heterotrimeric unit. These results suggested that the l-hydroxyproline pathway clearly evolved convergently in P. putida and P. aeruginosa. Pyr4H2C deaminase is a unique member of the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein family, and its activity was competitively inhibited by pyruvate, a common substrate for other dihydrodipicolinate synthase/N-acetylneuraminate lyase proteins. Furthermore, disruption of Pyr4H2C deaminase genes led to loss of growth on l-hydroxyproline (as well as d-hydroxyproline) but not l- and d-proline, indicating that this pathway is related only to l-hydroxyproline degradation, which is not linked to proline metabolism.     

Metabolic Mechanism of Mannan in a Ruminal Bacterium,Ruminococcus albus,Involving Two Mannoside Phosphorylases and Cellobiose 2-Epimerase: DISCOVERY OF A NEW CARBOHYDRATE PHOSPHORYLASE, β-1,4-MANNOOLIGOSACCHARIDE PHOSPHORYLASE*

Ruminococcus albus is a typical ruminal bacterium digesting cellulose and hemicellulose. Cellobiose 2-epimerase (CE; EC 5.1.3.11), which converts cellobiose to 4-O-β-d-glucosyl-d-mannose, is a particularly unique enzyme in R. albus, but its physiological function is unclear. Recently, a new metabolic pathway of mannan involving CE was postulated for another CE-producing bacterium, Bacteroides fragilis. In this pathway, β-1,4-mannobiose is epimerized to 4-O-β-d-mannosyl-d-glucose (Man-Glc) by CE, and Man-Glc is phosphorolyzed to α-d-mannosyl 1-phosphate (Man1P) and d-glucose by Man-Glc phosphorylase (MP; EC 2.4.1.281). Ruminococcus albus NE1 showed intracellular MP activity, and two MP isozymes, RaMP1 and RaMP2, were obtained from the cell-free extract. These enzymes were highly specific for the mannosyl residue at the non-reducing end of the substrate and catalyzed the phosphorolysis and synthesis of Man-Glc through a sequential Bi Bi mechanism. In a synthetic reaction, RaMP1 showed high activity only toward d-glucose and 6-deoxy-d-glucose in the presence of Man1P, whereas RaMP2 showed acceptor specificity significantly different from RaMP1. RaMP2 acted on d-glucose derivatives at the C2- and C3-positions, including deoxy- and deoxyfluoro-analogues and epimers, but not on those substituted at the C6-position. Furthermore, RaMP2 had high synthetic activity toward the following oligosaccharides: β-linked glucobioses, maltose, N,N′-diacetylchitobiose, and β-1,4-mannooligosaccharides. Particularly, β-1,4-mannooligosaccharides served as significantly better acceptor substrates for RaMP2 than d-glucose. In the phosphorolytic reactions, RaMP2 had weak activity toward β-1,4-mannobiose but efficiently degraded β-1,4-mannooligosaccharides longer than β-1,4-mannobiose. Consequently, RaMP2 is thought to catalyze the phosphorolysis of β-1,4-mannooligosaccharides longer than β-1,4-mannobiose to produce Man1P and β-1,4-mannobiose.     

An l-glucose Catabolic Pathway in Paracoccus Species 43P

An l-glucose-utilizing bacterium, Paracoccus sp. 43P, was isolated from soil by enrichment cultivation in a minimal medium containing l-glucose as the sole carbon source. In cell-free extracts from this bacterium, NAD⁺-dependent l-glucose dehydrogenase was detected as having sole activity toward l-glucose. This enzyme, LgdA, was purified, and the lgdA gene was found to be located in a cluster of putative inositol catabolic genes. LgdA showed similar dehydrogenase activity toward scyllo- and myo-inositols. l-Gluconate dehydrogenase activity was also detected in cell-free extracts, which represents the reaction product of LgdA activity toward l-glucose. Enzyme purification and gene cloning revealed that the corresponding gene resides in a nine-gene cluster, the lgn cluster, which may participate in aldonate incorporation and assimilation. Kinetic and reaction product analysis of each gene product in the cluster indicated that they sequentially metabolize l-gluconate to glycolytic intermediates, d-glyceraldehyde-3-phosphate, and pyruvate through reactions of C-5 epimerization by dehydrogenase/reductase, dehydration, phosphorylation, and aldolase reaction, using a pathway similar to l-galactonate catabolism in Escherichia coli. Gene disruption studies indicated that the identified genes are responsible for l-glucose catabolism.     

Alteration of intrinsic amounts of d-serine in the mice lacking serine racemase and d-amino acid oxidase

For elucidation of the regulation mechanisms of intrinsic amounts of d-serine (d-Ser) which modulates the neuro-transmission of N-methyl-d-aspartate receptors in the brain, mutant animals lacking serine racemase (SRR) and d-amino acid oxidase (DAO) were established, and the amounts of d-Ser in the tissues and physiological fluids were determined. d-Ser amounts in the frontal brain areas were drastically decreased followed by reduced SRR activity. On the other hand, a moderate but significant decrease in d-Ser amounts was observed in the cerebellum and spinal cord of SRR knock-out (SRR^?/?) mice compared with those of control mice, although the amounts of d-Ser in these tissues were low. The amounts of d-Ser in the brain and serum were not altered with aging. To clarify the uptake of exogenous d-Ser into the brain tissues, we have determined the d-Ser of SRR^?/? mice after oral administration of d-Ser for the first time, and a drastic increase in d-Ser amounts in all the tested tissues was observed. Because both DAO and SRR are present in some brain areas, we have established the double mutant mice lacking SRR and DAO for the first time, and the contribution of both enzymes to the intrinsic d-Ser amounts was investigated. In the frontal brain, most of the intrinsic d-Ser was biosynthesized by SRR. On the other hand, half of the d-Ser present in the hindbrain was derived from the biosynthesis by SRR. These results indicate that the regulation of intrinsic d-Ser amounts is different depending on the tissues and provide useful information for the development of treatments for neuronal diseases.     

Characterization of 5-Chloro-5-Deoxy-d-Ribose 1-Dehydrogenase in Chloroethylmalonyl Coenzyme A Biosynthesis: SUBSTRATE AND REACTION PROFILING*

SalM is a short-chain dehydrogenase/reductase enzyme from the marine actinomycete Salinispora tropica that is involved in the biosynthesis of chloroethylmalonyl-CoA, a novel halogenated polyketide synthase extender unit of the proteasome inhibitor salinosporamide A. SalM was heterologously overexpressed in Escherichia coli and characterized in vitro for its substrate specificity, kinetics, and reaction profile. A sensitive real-time ¹³C NMR assay was developed to visualize the oxidation of 5-chloro-5-deoxy-d-ribose to 5-chloro-5-deoxy-d-ribono-γ-lactone in an NAD⁺-dependent reaction, followed by spontaneous lactone hydrolysis to 5-chloro-5-deoxy-d-ribonate. Although short-chain dehydrogenase/reductase enzymes are widely regarded as metal-independent, a strong divalent metal cation dependence for Mg²⁺, Ca²⁺, or Mn²⁺ was observed with SalM. Oxidative activity was also measured with the alternative substrates d-erythrose and d-ribose, making SalM the first reported stereospecific non-phosphorylative ribose 1-dehydrogenase.