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1.
Djalal Meziane-Cherif Frederick A. Saul Ahmed Haouz Patrice Courvalin 《The Journal of biological chemistry》2012,287(45):37583-37592
d-Alanyl:d-lactate (d-Ala:d-Lac) and d-alanyl:d-serine ligases are key enzymes in vancomycin resistance of Gram-positive cocci. They catalyze a critical step in the synthesis of modified peptidoglycan precursors that are low binding affinity targets for vancomycin. The structure of the d-Ala:d-Lac ligase VanA led to the understanding of the molecular basis for its specificity, but that of d-Ala:d-Ser ligases had not been determined. We have investigated the enzymatic kinetics of the d-Ala:d-Ser ligase VanG from Enterococcus faecalis and solved its crystal structure in complex with ADP. The overall structure of VanG is similar to that of VanA but has significant differences mainly in the N-terminal and central domains. Based on reported mutagenesis data and comparison of the VanG and VanA structures, we show that residues Asp-243, Phe-252, and Arg-324 are molecular determinants for d-Ser selectivity. These residues are conserved in both enzymes and explain why VanA also displays d-Ala:d-Ser ligase activity, albeit with low catalytic efficiency in comparison with VanG. These observations suggest that d-Ala:d-Lac and d-Ala:d-Ser enzymes have evolved from a common ancestral d-Ala:d-X ligase. The crystal structure of VanG showed an unusual interaction between two dimers involving residues of the omega loop that are deeply anchored in the active site. We constructed an octapeptide mimicking the omega loop and found that it selectively inhibits VanG and VanA but not Staphylococcus aureus
d-Ala:d-Ala ligase. This study provides additional insight into the molecular evolution of d-Ala:d-X ligases and could contribute to the development of new structure-based inhibitors of vancomycin resistance enzymes. 相似文献
2.
Tian Xia Qi Han William V. Costanzo Yixuan Zhu Jeffrey L. Urbauer Mark A. Eiteman 《Applied and environmental microbiology》2015,81(10):3387-3394
Escherichia coli that is unable to metabolize d-glucose (with knockouts in ptsG, manZ, and glk) accumulates a small amount of d-glucose (yield of about 0.01 g/g) during growth on the pentoses d-xylose or l-arabinose as a sole carbon source. Additional knockouts in the zwf and pfkA genes, encoding, respectively, d-glucose-6-phosphate 1-dehydrogenase and 6-phosphofructokinase I (E. coli MEC143), increased accumulation to greater than 1 g/liter d-glucose and 100 mg/liter d-mannose from 5 g/liter d-xylose or l-arabinose. Knockouts of other genes associated with interconversions of d-glucose-phosphates demonstrate that d-glucose is formed primarily by the dephosphorylation of d-glucose-6-phosphate. Under controlled batch conditions with 20 g/liter d-xylose, MEC143 generated 4.4 g/liter d-glucose and 0.6 g/liter d-mannose. The results establish a direct link between pentoses and hexoses and provide a novel strategy to increase carbon backbone length from five to six carbons by directing flux through the pentose phosphate pathway. 相似文献
3.
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. 相似文献
4.
Partial Purification and Properties of l-Glutamine d-Fructose 6-Phosphate Amidotransferase from Phaseolus aureus 总被引:1,自引:1,他引:0
l-Glutamine d-fructose 6-phosphate amidotransferase (EC 2.6.1.16) was extracted and purified 600-fold by acetone fractionation and diethylaminoethyl cellulose column chromatography from mung bean seeds (Phaseolus aureus). The partially purified enzyme was highly specific for l-glutamine as an amide nitrogen donor, and l-asparagine could not replace it. The enzyme showed a pH optimum in the range of 6.2 to 6.7 in phosphate buffer. Km values of 3.8 mm and 0.5 mm were obtained for d-fructose 6-phosphate and l-glutamine, respectively. The enzyme was competitively inhibited with respect to d-fructose 6-phosphate by uridine diphosphate-N-acetyl-d-glucosamine which had a Ki value of 13 μm. Upon removal of l-glutamine and its replacement by d-fructose 6-phosphate and storage over liquid nitrogen, the enzyme was completely desensitized to inhibition by uridine diphosphate-N-acetyl-d-glucosamine. This indicates that the inhibitor site is distinct from the catalytic site and that uridine diphosphate-N-acetyl-d-glucosamine acts as a feedback inhibitor of the enzyme. 相似文献
5.
Amino Acid Isomerization in the Production of l-Phenylalanine from d-Phenylalanine by Bacteria
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To establish an advantageous method for the production of l-amino acids, microbial isomerization of d- and dl-amino acids to l-amino acids was studied. Screening experiments on a number of microorganisms showed that cell suspensions of Pseudomonas fluorescens and P. miyamizu were capable of isomerizing d- and dl-phenylalanines to l-phenylalanine. Various conditions suitable for isomerization by these organisms were investigated. Cells grown in a medium containing d-phenylalanine showed highest isomerization activity, and almost completely converted d- or dl-phenylalanine into l-phenylalanine within 24 to 48 hr of incubation. Enzymatic studies on this isomerizing system suggested that the isomerization of d- or dl-phenylalanine is not catalyzed by a single enzyme, “amino acid isomerase,” but the conversion proceeds by a two step system as follows: d-pheylalanine is oxidized to phenylpyruvic acid by d-amino acid oxidase, and the acid is converted to l-phenylalanine by transamination or reductive amination. 相似文献
6.
The Role of a d-Mannosyl-Lipid as an Intermediate in the Synthesis of Polysaccharide in Phaseolus aureus Seedlings 总被引:2,自引:1,他引:1
Particulate preparations from Phaseolus aureus produce a d-mannosyl-lipid when treated with GDP-d-mannose. This lipid complex appears to be an active d-mannose donor, and some investigators have proposed that its role might be an obligatory intermediate in mannan synthesis of higher plants. When the partially purified d-mannosyl-lipids, isotopically labeled in the d-mannose moiety, were treated with particulate enzymes under a variety of conditions, a negligible amount of material was produced that behaved as a polysaccharide. Endogenous, particle-bound d-mannosyl-14C-lipid prepared from P. aureus particles readily transferred d-mannose to GDP to yield GDP-d-mannose and was hydrolyzed to free d-mannose when treated briefly with 0.01 n HCl at 100 C. The d-mannosyl-lipid, therefore, exhibits active d-mannose transfer potential in its endogenous state. When endogenous glycosyl-lipid was incubated in the absence of GDP-d-mannose-14C, little or no polysaccharide was produced. It was, instead, slowly degraded to d-mannose. Addition of several different unlabeled sugar nucleotides had no effect on the results. Our studies to date, therefore, offer no evidence that the mannosyl-lipid is an obligatory precursor of polysaccharide. 相似文献
7.
Dominik Mojzita Merja Penttil? Peter Richard 《The Journal of biological chemistry》2010,285(31):23622-23628
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 Km for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm. 相似文献
8.
Sara A. Leiman Janine M. May Matthew D. Lebar Daniel Kahne Roberto Kolter Richard Losick 《Journal of bacteriology》2013,195(23):5391-5395
The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of d-leucine, d-methionine, d-tryptophan, and d-tyrosine and was reported to inhibit biofilm formation via the incorporation of these d-amino acids into the cell wall. Here, we show that l-amino acids were able to specifically reverse the inhibitory effects of their cognate d-amino acids. We also show that d-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding d-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of d-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of d-amino acids without losing the ability to incorporate at least one noncanonical d-amino acid, d-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of d-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis. 相似文献
9.
Takanori Kumagai Tomoki Ozawa Momoko Tanimoto Masafumi Noda Yasuyuki Matoba Masanori Sugiyama 《Applied and environmental microbiology》2015,81(22):7881-7887
Previously, we successfully cloned a d-cycloserine (d-CS) biosynthetic gene cluster consisting of 10 open reading frames (designated dcsA to dcsJ) from d-CS-producing Streptomyces lavendulae ATCC 11924. In this study, we put four d-CS biosynthetic genes (dcsC, dcsD, dcsE, and dcsG) in tandem under the control of the T7 promoter in an Escherichia coli host. SDS-PAGE analysis demonstrated that the 4 gene products were simultaneously expressed in host cells. When l-serine and hydroxyurea (HU), the precursors of d-CS, were incubated together with the E. coli resting cell suspension, the cells produced significant amounts of d-CS (350 ± 20 μM). To increase the productivity of d-CS, the dcsJ gene, which might be responsible for the d-CS excretion, was connected downstream of the four genes. The E. coli resting cells harboring the five genes produced d-CS at 660 ± 31 μM. The dcsD gene product, DcsD, forms O-ureido-l-serine from O-acetyl-l-serine (OAS) and HU, which are intermediates in d-CS biosynthesis. DcsD also catalyzes the formation of l-cysteine from OAS and H2S. To repress the side catalytic activity of DcsD, the E. coli chromosomal cysJ and cysK genes, encoding the sulfite reductase α subunit and OAS sulfhydrylase, respectively, were disrupted. When resting cells of the double-knockout mutant harboring the four d-CS biosynthetic genes, together with dcsJ, were incubated with l-serine and HU, the d-CS production was 980 ± 57 μM, which is comparable to that of d-CS-producing S. lavendulae ATCC 11924 (930 ± 36 μM). 相似文献
10.
Krzysztof Regulski Pascal Courtin Saulius Kulakauskas Marie-Pierre Chapot-Chartier 《The Journal of biological chemistry》2013,288(28):20416-20426
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. 相似文献
11.
This study presents evidence for a new enzyme, d-ribose-5-P reductase, which catalyzes the reaction: d-ribose-5-P + NADPH + H+ → d-ribitol-5-P + NADP+. The enzyme was isolated from Adonis vernalis L. leaves in 38% yield and was purified 71-fold. The reductase was NADPH specific and had a pH optimum in the range of 5.5 to 6.0. The Michaelis constant value for d-ribose-5-P reduction was 1.35 millimolar. The enzyme also reduced d-erythrose-4-P, d-erythrose, dl-glyceraldehyde, and the aromatic aldehyde 3-pyridinecarboxaldehyde. Hexoses, hexose phosphates, pentoses, and dihydroxyacetone did not serve as substrates. d-Ribose-5-P reductase is distinct from the other known ribitol synthesizing enzymes detected in bacteria and yeast, and may be responsible for ribitol synthesis in Adonis vernalis. 相似文献
12.
DdlN from Vancomycin-Producing Amycolatopsis orientalis C329.2 Is a VanA Homologue with d-Alanyl-d-Lactate Ligase Activity
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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 ( A4793414). 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.
Open in a separate windowOpen in a separate windowFIG. 1Purification of DdlN from E. coli BL21 (DE3)/pETDdlN. Proteins were separated on an SDS–11% polyacrylamide gel and stained with Coomassie blue. Lane 1, molecular mass markers (masses are noted at the left in kilodaltons); lane 2, whole-cell lysate; lane 3, ammonium sulfate fraction (20 to 50% saturation); lane 4, Sephacryl S200; lane 5, Q Sepharose; lane 6, phenyl Superose.The amino acid substrate specificity of DdlN was assessed by incubation of 14C-d-Ala with all 20 common amino acids in the d configuration. Purified DdlN catalyzed the synthesis of d-Ala-d-Ala in addition to that of several other mixed dipeptides, including d-Ala-d-Met and d-Ala-d-Phe (Fig. (Fig.2).2). Thus, DdlN exhibits a substrate specificity which is similar to that of VanA (4), with the capacity to synthesize not only d-Ala-d-Ala but also mixed dipeptides with bulky side chains in the C-terminal position.Open in a separate windowFIG. 2Substrate specificity of DdlN. Autoradiogram from thin-layer chromatography analysis of DdlN substrate specificity. All reaction mixtures contained 2.5 mM d-Ala and 1 mM ATP, and the radiolabel was 14C-d-Ala, except where noted. Lane 1, d-Ala; lane 2, d-Lac with 14C-d-Lac label; lane 3, d,l-methionine; lane 4, dl-phenylalanine; lane 5, d-Hbut; lane 6, d-hydroxyvalerate. Letters indicate the following: A, d-Ala-d-Lac; B, d-Lac; C, d-Ala-d-Met; D, d-Ala-d-Phe; E, d-Ala-d-Hbut; F, d-Ala-d-hydroxyvalerate.Importantly, DdlN is a depsipeptide synthase with the ability to synthesize d-Ala-d-Lac, d-Ala-d-hydroxybutyrate (Hbut), and d-Ala-d-hydroxyvalerate (Fig. (Fig.2).2). However, unlike VanA (5), d-hydroxycaproate and d-phenyllactate are not substrates (not shown). Thus, DdlN is a broad-spectrum d-Ala-d-X ligase with depsipeptide synthase activity.
Open in a separate windowa Determined in the presence of 10 mM d-Lac. b Data from reference 16. c Data from reference 5. DdlN showed good d-Ala-d-Ala ligase activity but with a very high and physiologically questionable Km2 (21 mM). On the other hand, d-Ala-d-Lac synthesis was excellent, with a 4-fold decrease in kcat, compared to d-Ala-d-Ala synthesis, which was offset by a 52-fold drop in Km that resulted in a >12-fold increase in specificity (kcat/Km2). d-Hbut was also a good substrate, with a kcat/Km2 comparable to that of d-Ala.Steady-state kinetic parameters for d-Ala-d-X formation showed trends similar to those found with both VanA and DdlN. For example, the kcat values between VanA and DdlN were virtually the same for most substrates. There were significant differences, however. For instance, while the Km2 values for d-Ala were very high for all three enzymes, DdlN does have greater affinity for d-Ala, with a 1.8- and 7.9-fold lower Km2 than those of VanA and DdlM, respectively. Additionally, the Km2 for d-Lac was 17.8- and 2.7-fold lower than those for VanA and DdlM. Thus, DdlN has a more restrictive specificity for the C-terminal residue than VanA, which is compensated for by a higher affinity for the critical substrate d-Lac.
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)/pETDdlNSample | Protein (mg) | Activity (nmol/min) | Sp act (nmol/ min/mg) | Recovery (%) | Purification (fold) |
---|---|---|---|---|---|
Lysate | 124 | 843 | 6.82 | 100 | |
Ammonium sulfate (20–50% saturation) | 67.6 | 780 | 11.5 | 92 | 1.7 |
Sephacryl S200 | 11.6 | 825 | 71.4 | 98 | 11 |
Q Sepharose | 2.8 | 742 | 265 | 88 | 39 |
Phenyl Superose | 0.4 | 299 | 748 | 35 | 110 |
Characterization of d-Ala-d-X ligase activity.
Following the initial assessment of the specificity of the enzyme, several substrates were selected for quantitative analysis by evaluation of their steady-state kinetic parameters (Table (Table2).2). DdlN has two amino acid (or hydroxy acid) Km values. Steady-state kinetic plots indicated that, like other dd-ligases, the N-terminal Km (Km1) was significantly lower (higher specificity) than the C-terminal Km (Km2). Since the former value is expected to be independent of the C-terminal substrate, only Km2 values were determined and are reported here.TABLE 2
Characterization of steady-state parameters of DdlN and VanALigase | Substrate | Km2 (mM) | kcat (min−1) | kcat/Km2 (M−1 s−1) |
---|---|---|---|---|
DdlN | d-Ala | 21 ± 2 | 229 ± 7 | 1.8 × 102 |
d-Lac | 0.4 ± 0.05 | 55 ± 1 | 2.3 × 103 | |
d-Hbut | 2.5 ± 0.3 | 32 ± 2 | 2.1 × 102 | |
ATPa | 1.2 ± 0.2 | 71 ± 5 | 0.98 × 102 | |
DdlMb | d-Ala | 166 ± 27 | ||
d-Lac | 1.08 ± 0.10 | |||
VanAc | d-Ala | 38 | 295 | 1.3 × 102 |
d-Lac | 7.1 | 94 | 2.2 × 102 | |
d-Hbut | 0.60 | 108 | 3.0 × 103 |
pH dependence of peptide versus that of depsipeptide synthesis activity.
The partitioning of the syntheses of d-Ala-d-Ala and d-Ala-d-Hbut in VanA and other depsipeptide-competent dd-ligases has been shown to be pH dependent (17). Determination of the pH dependence of DdlN in synthesizing peptide versus depsipeptide (Fig. (Fig.3)3) directly paralleled the results obtained with VanA in similar experiments. At lower pHs (<7), d-Ala-d-Hbut synthesis predominates and is exclusive at a pH of <6 (Fig. (Fig.3).3). At pH 7.5, levels of synthesis of d-Ala-d-Hbut and d-Ala-d-Ala are relatively equal, while at a pH greater than 8, the capacity to synthesize peptide overtakes the capacity to synthesize depsipeptide, although the latter is never abolished. Open in a separate windowFIG. 3pH dependence of partitioning of the syntheses of peptide and depsipeptide by DdlN. (A) Autoradiogram of a thin-layer chromatography separation of the products of reaction mixtures containing 14C-D-Ala, unlabeled D-Ala, and d-Hbut. (B) Quantification of reaction products following phosphorimage analysis. Filled circles, D-Ala-d-Hbut; open circles, D-Ala-D-Ala.The partitioning of the formation of peptide versus depsipeptide as a function of pH by DdlM is comparable to that by VanA and depsipeptide-competent mutants of DdlB (17), which show essentially exclusively depsipeptide formation at lower pHs and increasing peptide formation as the pH increases. This implies a potential role for the protonated ammonium group of d-Ala2 in second-substrate recognition and suggests a mechanism for the discrimination between d-Ala and d-Lac at physiologic pH. The structural basis for this distinction remains obscure for DdlB and VanA or DdlN.Concluding remarks.
Resistance to vancomycin and other glycopeptides is mediated through the synthesis of a peptidoglycan which does not terminate with the canonical d-Ala-d-Ala dipeptide. Thus, enterococci which exhibit the VanC phenotype, which consists of low-level, noninducible resistance to vancomycin only, have peptidoglycan terminating in d-Ala-d-Ser (19). On the other hand, bacteria which are constitutively resistant to high concentrations of glycopeptides, such as lactic acid bacteria and VRE exhibiting the VanA or VanB phenotype (high-level inducible resistance to vancomycin), incorporate the depsipeptide d-Ala-d-Lac into their cell walls (2, 12, 13). The enzymes responsible for the intracellular synthesis of d-Ala-d-Lac not surprisingly have significant amino acid sequence similarity with d-Ala-d-Ala ligases, which are responsible for d-Ala-d-Ala synthesis in all bacteria with a cell wall (9).The d-Ala-d-Lac synthases can be subdivided into two groups based on sequence homology: those found in the constitutively resistant lactic acid bacteria and those found in glycopeptide-producing organisms and VanA or VanB VRE (9, 14). The former have more similarity with exclusive d-Ala-d-Ala ligases. Indeed, single point mutations in d-Ala-d-Ala ligases which yield sequences more similar to those of lactic acid bacterium d-Ala-d-Lac ligases are sufficient to induce significant depsipeptide synthase activity in these enzymes (17). Similarly, mutational studies of the d-Ala-d-Lac ligase from Leuconostoc mesenteroides have demonstrated that the converse also holds (18). On the other hand, the molecular basis for depsipeptide synthesis by the VanA or VanB ligases is unknown, in large part due to the lack of protein structural information on which to base mutational studies, unlike the situation with d-Ala-d-Ala ligases, where the E. coli DdlB structure serves as a template for mechanistic research (11).Significantly, a major difference in the VanA or VanB ligases and other dd-ligases lies in the amino acid sequence of the ω-loop region, which closes off the active site of DdlB (11) and has been shown to contribute amino acid residues with the capacity to control the syntheses of d-Ala-d-Ala and d-Ala-d-Lac, notably, Tyr216 (17, 18). Until recently, the VanA and VanB ligases were exceptional in amino acid structure and had no known homologues. The sequencing of resistance genes from glycopeptide-producing bacteria has uncovered enzymes with >60% homology to VanA or VanB and which are virtually superimposable in the critical ω-loop region (14, 15). One of these, DdlM from S. toyocaensis NRRL 15009, has been shown to have d-Ala-d-Lac ligase ability (15, 16), although no rigorous analysis of this activity has been performed. The results presented here demonstrate that DdlN from the vancomycin producer A. orientalis C329.2 not only is a d-Ala-d-Lac ligase but also has significant functional homology with VanA. It is not known at present if, like S. toyocaensis NRRL 15009 (16), A. orientalis C329.2 also possess a d-Ala-d-Ala-exclusive ligase, though the presence of a vanX gene (14) suggests that it may.These studies demonstrate that DdlN cloned from a vancomycin-producing bacterium is a d-Ala-d-Lac ligase which has not only amino acid sequence homology with the dd-ligases from VRE but also functional homology. Thus, VanA, VanB, DdlN, and DdlM have likely evolved from similar origins. The fact that a vanH-vanA-vanX gene cluster can be found in other glycopeptide producers as well (14) suggests that the genes now found in VRE may have originated in glycopeptide-producing bacteria. Our finding that overexpressed, purified, DdlN shows many enzymatic characteristics similar (though not identical) to those of VanA suggests that the genes from glycopeptide-producing bacteria can be important in elucidating biochemical and protein structural aspects of the VRE proteins. 相似文献13.
Dominik Mojzita Marilyn Wiebe Satu Hilditch Harry Boer Merja Penttil? Peter Richard 《Applied and environmental microbiology》2010,76(1):169-175
d-Galacturonic acid can be obtained by hydrolyzing pectin, which is an abundant and low value raw material. By means of metabolic engineering, we constructed fungal strains for the conversion of d-galacturonate to meso-galactarate (mucate). Galactarate has applications in food, cosmetics, and pharmaceuticals and as a platform chemical. In fungi d-galacturonate is catabolized through a reductive pathway with a d-galacturonate reductase as the first enzyme. Deleting the corresponding gene in the fungi Hypocrea jecorina and Aspergillus niger resulted in strains unable to grow on d-galacturonate. The genes of the pathway for d-galacturonate catabolism were upregulated in the presence of d-galacturonate in A. niger, even when the gene for d-galacturonate reductase was deleted, indicating that d-galacturonate itself is an inducer for the pathway. A bacterial gene coding for a d-galacturonate dehydrogenase catalyzing the NAD-dependent oxidation of d-galacturonate to galactarate was introduced to both strains with disrupted d-galacturonate catabolism. Both strains converted d-galacturonate to galactarate. The resulting H. jecorina strain produced galactarate at high yield. The A. niger strain regained the ability to grow on d-galacturonate when the d-galacturonate dehydrogenase was introduced, suggesting that it has a pathway for galactarate catabolism.d-Galacturonate is the main component of pectin, an abundant and cheap raw material. Sugar beet pulp and citrus peel are both rich in pectin residues. At present, these residues are mainly used as cattle feed. However, since energy-consuming drying and pelletizing of the residues is required to prevent them from rotting, it is not always economical to process the residues, and it is desirable to find alternative uses.Various microbes which live on decaying plant material have the ability to catabolize d-galacturonate using various, completely different pathways (19). Eukaryotic microorganisms use a reductive pathway in which d-galacturonate is first reduced to l-galactonate by an NAD(P)H-dependent reductase (12, 17). In the following steps a dehydratase, aldolase, and reductase convert the l-galactonate to pyruvate and glycerol (9, 11, 14).In Hypocrea jecorina (anamorph Trichoderma reesei) the gar1 gene codes for a strictly NADPH-dependent d-galacturonate reductase. In Aspergillus niger a homologue gene sequence, gar2, exists; however, a different gene, gaaA, is upregulated during growth on d-galacturonate containing medium (16). The gaaA codes for a d-galacturonate reductase with different kinetic properties than the H. jecorina enzyme, having a higher affinity toward d-galacturonate and using either NADH or NADPH as cofactor. It is not known whether gar2 codes for an active protein.Some bacteria, such as Agrobacterium tumefaciens or Pseudomonas syringae, have an oxidative pathway for d-galacturonate catabolism. In this pathway d-galacturonate is first oxidized to meso-galactarate (mucate) by an NAD-utilizing d-galacturonate dehydrogenase. Galactarate is then converted in the following steps to α-ketoglutarate. This route is sometimes called the α-ketoglutarate pathway (20). Galactarate can also be catabolized through the glycerate pathway (20). The products of this pathway are pyruvate and d-glycerate. These pathways have been described in prokaryotes, and it is not certain whether similar pathways also exist in fungi, some of which are able to metabolize galactarate.d-Galacturonate dehydrogenase (EC 1.1.1.203) has been described in Agrobacterium tumefaciens and in Pseudomonas syringae, and the enzymes from these organisms have been purified and characterized (3, 6, 22). Recently, the corresponding genes were also identified (4, 24). Both enzymes are specific for NAD as a cofactor but are not specific for the substrate. They oxidize d-galacturonate and d-glucuronate to meso-galactarate (mucate) and d-glucarate (saccharate), respectively. The reaction product is probably the hexaro-lactone which spontaneously hydrolyzes. The reverse reaction can only be observed at acidic pH where some of the galactarate is in the lactone form (22).We describe here strains of filamentous fungi that have been genetically engineered to produce galactarate by disruption of d-galacturonate reductase and expression of d-galacturonate dehydrogenase (Fig. (Fig.1).1). Galactarate is currently commercially produced from d-galactose by oxidation with nitric acid (1) or from d-galacturonate by electrolytic oxidation (8). Oxidation with nitric acid is expensive and produces toxic wastes. Galactarate is used as a chelator and in skin care products. It was formerly used as a leavening agent in self-rising flour (2) and has potential applications in polymer synthesis (10) and as a platform chemical (for a review, see reference 13).Open in a separate windowFIG. 1.Engineering the d-galacturonic acid pathway in fungi. Deletion of the gene encoding d-galacturonate reductase resulted in strains unable to utilize d-galacturonic acid as a carbon source. The expression of a bacterial udh gene, encoding an NAD-dependent d-galacturonate dehydrogenase, resulted in fungal strains, which were able to oxidize d-galacturonic acid to meso-galactaric acid (mucic acid). d-Galacturonate dehydrogenase forms a galactaro-lactone which spontaneously hydrolyzes. 相似文献
14.
In Vitro Analysis of the H-Hexose Symporter on the Plasma Membrane of Sugarbeets (Beta vulgaris L.)
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The mechanism of hexose transport into plasma membrane vesicles isolated from mature sugarbeet leaves (Beta vulgaris L.) was investigated. The initial rate of glucose uptake into the vesicles was stimulated approximately fivefold by imposing a transmembrane pH gradient (ΔpH), alkaline inside, and approximately fourfold by a negative membrane potential (ΔΨ), generated as a K+-diffusion potential, negative inside. The -fold stimulation was directly related to the relative ΔpH or ΔΨ gradient imposed, which were determined by the uptake of acetate or tetraphenylphosphonium, respectively. ΔΨ- and ΔpH-dependent glucose uptake showed saturation kinetics with a Km of 286 micromolar for glucose. Other hexose molecules (e.g. 2-deoxy-d-glucose, 3-O-methyl-d-glucose, and d-mannose) were also accumulated into plasma membrane vesicles in a ΔpH-dependent manner. Inhibition constants of a number of compounds for glucose uptake were determined. Effective inhibitors of glucose uptake included: 3-O-methyl-d-glucose, 5-thio-d-glucose, d-fructose, d-galactose, and d-mannose, but not 1-O-methyl-d-glucose, d- and l-xylose, l-glucose, d-ribose, and l-sorbose. Under all conditions of proton motive force magnitude and glucose and sucrose concentration tested, there was no effect of sucrose on glucose uptake. Thus, hexose transport on the sugarbeet leaf plasma membrane was by a H+-hexose symporter, and the carrier and possibly the energy source were not shared by the plasma membrane H+-sucrose symporter. 相似文献
15.
The oxidation of d- and l-glycerate by rat liver 总被引:1,自引:1,他引:0
1. The interconversion of hydroxypyruvate and l-glycerate in the presence of NAD and rat-liver l-lactate dehydrogenase has been demonstrated. Michaelis constants for these substrates together with an equilibrium constant have been determined and compared with those for pyruvate and l-lactate. 2. The presence of d-glycerate dehydrogenase in rat liver has been confirmed and the enzyme has been purified 16–20-fold from the supernatant fraction of a homogenate, when it is free of l-lactate dehydrogenase, with a 23–29% recovery. The enzyme catalyses the interconversion of hydroxypyruvate and d-glycerate in the presence of either NAD or NADP with almost equal efficiency. d-Glycerate dehydrogenase also catalyses the reduction of glyoxylate, but is distinct from l-lactate dehydrogenase in that it fails to act on pyruvate, d-lactate or l-lactate. The enzyme is strongly dependent on free thiol groups, as shown by inhibition with p-chloromercuribenzoate, and in the presence of sodium chloride the reduction of hydroxypyruvate is activated. Michaelis constants for these substrates of d-glycerate dehydrogenase and an equilibrium constant for the NAD-catalysed reaction have been calculated. 3. An explanation for the lowered Vmax. with d-glycerate as compared with dl-glycerate for the rabbit-kidney d-α-hydroxy acid dehydrogenase has been proposed. 相似文献
16.
Ramesh Prasad Pandey Prakash Parajuli Ju Yong Shin Jisun Lee Seul Lee Young-Soo Hong Yong Il Park Joong Su Kim Jae Kyung Sohng 《Applied and environmental microbiology》2014,80(23):7235-7243
A UDP glucosyltransferase from Bacillus licheniformis was overexpressed, purified, and incubated with nucleotide diphosphate (NDP) d- and l-sugars to produce glucose, galactose, 2-deoxyglucose, viosamine, rhamnose, and fucose sugar-conjugated resveratrol glycosides. Significantly higher (90%) bioconversion of resveratrol was achieved with α-d-glucose as the sugar donor to produce four different glucosides of resveratrol: resveratrol 3-O-β-d-glucoside, resveratrol 4′-O-β-d-glucoside, resveratrol 3,5-O-β-d-diglucoside, and resveratrol 3,5,4′-O-β-d-triglucoside. The conversion rates and numbers of products formed were found to vary with the other NDP sugar donors. Resveratrol 3-O-β-d-2-deoxyglucoside and resveratrol 3,5-O-β-d-di-2-deoxyglucoside were found to be produced using TDP-2-deoxyglucose as a donor; however, the monoglycosides resveratrol 4′-O-β-d-galactoside, resveratrol 4′-O-β-d-viosaminoside, resveratrol 3-O-β-l-rhamnoside, and resveratrol 3-O-β-l-fucoside were produced from the respective sugar donors. Altogether, 10 diverse glycoside derivatives of the medically important resveratrol were generated, demonstrating the capacity of YjiC to produce structurally diverse resveratrol glycosides. 相似文献
17.
Binbin Sheng Jing Xu Yingxin Zhang Tianyi Jiang Sisi Deng Jian Kong Chao Gao Cuiqing Ma Ping Xu 《Applied and environmental microbiology》2015,81(12):4098-4110
d-Lactate was identified as one of the few available organic acids that supported the growth of Gluconobacter oxydans 621H in this study. Interestingly, the strain used d-lactate as an energy source but not as a carbon source, unlike other lactate-utilizing bacteria. The enzymatic basis for the growth of G. oxydans 621H on d-lactate was therefore investigated. Although two putative NAD-independent d-lactate dehydrogenases, GOX1253 and GOX2071, were capable of oxidizing d-lactate, GOX1253 was the only enzyme able to support the d-lactate-driven growth of the strain. GOX1253 was characterized as a membrane-bound dehydrogenase with high activity toward d-lactate, while GOX2071 was characterized as a soluble oxidase with broad substrate specificity toward d-2-hydroxy acids. The latter used molecular oxygen as a direct electron acceptor, a feature that has not been reported previously in d-lactate-oxidizing enzymes. This study not only clarifies the mechanism for the growth of G. oxydans on d-lactate, but also provides new insights for applications of the important industrial microbe and the novel d-lactate oxidase. 相似文献
18.
Shewanella oneidensis couples oxidation of lactate to respiration of many substrates. Here we report that llpR (l-lactate-positive regulator, SO_3460) encodes a positive regulator of l-lactate utilization distinct from previously studied regulators. We also demonstrate d-lactate inhibition of l-lactate utilization in S. oneidensis, resulting in preferential utilization of the d isomer. 相似文献
19.
Joosu Kuivanen Dominik Mojzita Yanming Wang Satu Hilditch Merja Penttil? Peter Richard Marilyn G. Wiebe 《Applied and environmental microbiology》2012,78(24):8676-8683
d-Galacturonic acid, the main monomer of pectin, is an attractive substrate for bioconversions, since pectin-rich biomass is abundantly available and pectin is easily hydrolyzed. l-Galactonic acid is an intermediate in the eukaryotic pathway for d-galacturonic acid catabolism, but extracellular accumulation of l-galactonic acid has not been reported. By deleting the gene encoding l-galactonic acid dehydratase (lgd1 or gaaB) in two filamentous fungi, strains were obtained that converted d-galacturonic acid to l-galactonic acid. Both Trichoderma reesei Δlgd1 and Aspergillus niger ΔgaaB strains produced l-galactonate at yields of 0.6 to 0.9 g per g of substrate consumed. Although T. reesei Δlgd1 could produce l-galactonate at pH 5.5, a lower pH was necessary for A. niger ΔgaaB. Provision of a cosubstrate improved the production rate and titer in both strains. Intracellular accumulation of l-galactonate (40 to 70 mg g biomass−1) suggested that export may be limiting. Deletion of the l-galactonate dehydratase from A. niger was found to delay induction of d-galacturonate reductase and overexpression of the reductase improved initial production rates. Deletion of the l-galactonate dehydratase from A. niger also delayed or prevented induction of the putative d-galacturonate transporter An14g04280. In addition, A. niger ΔgaaB produced l-galactonate from polygalacturonate as efficiently as from the monomer. 相似文献
20.
Soo-Jin Yeom Jung-Hwan Ji Nam-Hee Kim Chang-Su Park Deok-Kun Oh 《Applied and environmental microbiology》2009,75(14):4705-4710
The uncharacterized gene previously proposed as a mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme was observed at pH 7.5 and 40°C in the presence of 0.5 mM Co2+. The isomerization activity was specific for aldose substrates possessing hydroxyl groups oriented in the same direction at the C-2 and C-3 positions, such as the d and l forms of ribose, lyxose, talose, mannose, and allose. The enzyme exhibited the highest activity for l-ribulose among all pentoses and hexoses. Thus, l-ribose, as a potential starting material for many l-nucleoside-based pharmaceutical compounds, was produced at 213 g/liter from 300-g/liter l-ribulose by mannose-6-phosphate isomerase at 40°C for 3 h, with a conversion yield of 71% and a volumetric productivity of 71 g liter−1 h−1.l-Ribose is a potential starting material for the synthesis of many l-nucleoside-based pharmaceutical compounds, and it is not abundant in nature (5, 19). l-Ribose has been produced mainly by chemical synthesis from l-arabinose, l-xylose, d-glucose, d-galactose, d-ribose, or d-mannono-1,4-lactone (2, 17, 23). Biological l-ribose manufacture has been investigated using ribitol or l-ribulose. Recently, l-ribose was produced from ribitol by a recombinant Escherichia coli containing an NAD-dependent mannitol-1-dehydrogenase (MDH) with a 55% conversion yield when 100 g/liter ribitol was used in a 72-h fermentation (18). However, the volumetric productivity of l-ribose in the fermentation is 28-fold lower than that of the chemical method synthesized from l-arabinose (8). l-Ribulose has been biochemically converted from l-ribose using an l-ribose isomerase from an Acinetobacter sp. (9), an l-arabinose isomerase mutant from Escherichia coli (4), a d-xylose isomerase mutant from Actinoplanes missouriensis (14), and a d-lyxose isomerase from Cohnella laeviribosi (3), indicating that l-ribose can be produced from l-ribulose by these enzymes. However, the enzymatic production of l-ribulose is slow, and the enzymatic production of l-ribose from l-ribulose has been not reported.Sugar phosphate isomerases, such as ribose-5-phosphate isomerase, glucose-6-phosphate isomerase, and galactose-6-phosphate isomerase, work as general aldose-ketose isomerases and are useful tools for producing rare sugars, because they convert the substrate sugar phosphates and the substrate sugars without phosphate to have a similar configuration (11, 12, 21, 22). l-Ribose isomerase from an Acinetobacter sp. (9) and d-lyxose isomerase from C. laeviribosi (3) had activity with l-ribose, d-lyxose, and d-mannose. Thus, we can apply mannose-6-phosphate (EC 5.3.1.8) isomerase to the production of l-ribose, because there are no sugar phosphate isomerases relating to l-ribose and d-lyxose. The production of the expensive sugar l-ribose (bulk price, $1,000/kg) from the rare sugar l-ribulose by mannose-6-phosphate isomerase may prove to be a valuable industrial process, because we have produced l-ribulose from the cheap sugar l-arabinose (bulk price, $50/kg) using the l-arabinose isomerase from Geobacillus thermodenitrificans (20) (Fig. (Fig.11).Open in a separate windowFIG. 1.Schematic representation for the production of l-ribulose from l-arabinose by G. thermodenitrificans l-arabinose isomerase and the production of l-ribose from l-ribulose by B. subtilis mannose-6-phosphate isomerase.In this study, the gene encoding mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in E. coli. The substrate specificity of the recombinant enzyme for various aldoses and ketoses was investigated, and l-ribulose exhibited the highest activity among all pentoses and hexoses. Therefore, mannose-6-phosphate isomerase was applied to the production of l-ribose from l-ribulose. 相似文献