Gene cloning and characterization of glycine oxidase from newly isolated <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain R5

The gene encoding the glycine oxidase from Bacillus subtilis strain R5 (goxR) was cloned and expressed in Escherichia coli. The gene consisted of 1,110 nucleotides that encoded a protein (GoxR) of 369 amino acid residues with a molecular mass of 40,761 Da. The GoxR exhibited 98.6% identity with glycine oxidase from B. subtilis strain 168. Gene expression and purification of the recombinant GoxR were performed. The recombinant GoxR existed in a homotetramer form. The recombinant protein effectively catalyzed the oxidation of glycine and d-alanine. The specific activity of the purified recombinant GoxR was 0.96 U/mg when glycine was used as a substrate and 1.0 U/mg when d-alanine was substrate. The enzyme displayed its highest activity at pH 8.0 and at a temperature of 50°C. The activation energy of the reaction catalyzed by the enzyme was calculated to be 26 kJ/mol. The enzyme activity was significantly inhibited in the presence of organic solvents. No enhancement of enzyme activity was observed in the presence of metal cations. The experimental results presented in this study demonstrate that the enzyme was a bonafide glycine oxidase.     

Characterization of <Emphasis Type="SmallCaps">d</Emphasis>-tagatose-3-epimerase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> that converts <Emphasis Type="SmallCaps">d</Emphasis>-fructose into <Emphasis Type="SmallCaps">d-</Emphasis>psicose

A non-characterized gene, previously proposed as the d-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with d-fructose and decreased for other substrates in the order: d-tagatose, d-psicose, d-ribulose, d-xylulose and d-sorbose. Its activity was maximal at pH 9 and 40°C while being enhanced by Mn²⁺. At pH 9 and 40°C, 118 g d-psicose l⁻¹ was produced from 700 g d-fructose l⁻¹ after 3 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Detection of a single,metal-independent aminopeptidase activity in polymyxin-extracts from Escherichia coli B

Summary The supernatant of Escherichia coli B autolysed in the presence of polymyxin B contains a single, metal-independent aminopeptidase activity (E.C.-group 3.4.1). The enzyme cleaves the 4-nitroanilides of L-alanine, L-lysine, L-leucine, glycine, and weakly L-phenylalanine. The corresponding N-acetyl-L-alanine-, L-glutamic acid- and L-cysteine-derivatives are not attacked.Dedicated to Dr. Hans Poschenrieder on the occasion of his 75th birthday.     

Evolution of structure and substrate specificity ind-alanine:d-Alanine ligases and related enzymes

Thed-alanine:d-alanine-ligase-related enzymes can have three preferential substrate specificities. Usually, these enzymes synthesized-alanyl-d-alanine. In vancomycin-resistant Gram-positive bacteria, structurally related enzymes synthesized-alanyl-d-lactate or Dalanyl-d-serine. The sequence of internal fragments of eight structurald-alanine:d-alanine ligase genes from enterococci has been determined. Alignment of the deduced amino acid sequences with those of other related enzymes from Gram-negative and Gram-positive bacteria revealed the presence of four distinct sequence patterns in the putative substrate-binding sites, each correlating with specificity to a particular substrate (d-alanine:d-lactate ligases exhibited two patterns). Phylogenetic analysis showed different clusters. The enterococcal subtree was largely superimposable on that derived from 16S rRNA sequences. In lactic acid bacteria, structural divergence due to differences in substrate specificity was observed. Glycopeptide resistance proteins VanA and VanB, the VanC-type ligases, and Dd1A and DdlB from enteric bacteria andHaemophilus influenzae constituted separate clusters. Correspondence to: P. Courvalin     

Characterization of a recombinant cellobiose 2-epimerase from <Emphasis Type="BoldItalic">Caldicellulosiruptor saccharolyticus</Emphasis> and its application in the production of mannose from glucose

A putative N-acyl-d-glucosamine 2-epimerase from Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was identified as a cellobiose 2-epimerase by the analysis of the activity for substrates, acid-hydrolyzed products, and amino acid sequence. The cellobiose 2-epimerase was purified with a specific activity of 35 nmol min^–1 mg^–1 for d-glucose with a 47-kDa monomer. The epimerization activity for d-glucose was maximal at pH 7.5 and 75°C. The half-lives of the enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 142, 71, 35, 18, and 4.6 h, respectively. The enzyme catalyzed the epimerization reactions of the aldoses harboring hydroxyl groups oriented in the right-hand configuration at the C2 position and the left-hand configuration at the C3 position, such as d-glucose, d-xylose, l-altrose, l-idose, and l-arabinose, to their C2 epimers, such as d-mannose, d-lyxose, l-allose, l-gulose, and l-ribose, respectively. The enzyme catalyzed also the isomerization reactions. The enzyme exhibited the highest activity for mannose among monosaccharides. Thus, mannose at 75 g l^–1 and fructose at 47.5 g l^–1 were produced from 500 g l^–1 glucose at pH 7.5 and 75°C over 3 h by the enzyme.     

Characterization of raffinose synthase from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L. var. Nipponbare)

The putative raffinose synthase gene from rice was cloned and expressed in Escherichia coli. The enzyme displayed an optimum activity at 45°C and pH 7.0, and a sulfhydryl group was required for its activity. The enzyme was specific for galactinol and p-nitrophenyl-α-d-galactoside as galactosyl donors, and sucrose, lactose, 4−β-galactobiose, N-acetyl-d-lactosamine, trehalose and lacto-N-biose were recognized as galactosyl acceptors.     

Removal of <Emphasis Type="SmallCaps">l</Emphasis>-alanine from the production of <Emphasis Type="SmallCaps">l</Emphasis>-2-aminobutyric acid by introduction of alanine racemase and <Emphasis Type="SmallCaps">d</Emphasis>-amino acid oxidase

l-2-Aminobutyric acid can be synthesized in a transamination reaction from l-threonine and l-aspartic acid as substrates by the action of threonine deaminase and aromatic aminotransferase, but the by-product l-alanine was produced simultaneously. A small amount of l-alanine increased the complexity of the l-2-aminobutyric acid recovery process because of their extreme similarity in physical and chemical properties. Acetolactate synthase has been introduced to remove the pyruvate intermediate for reducing the l-alanine concentration partially. To eliminate the remnant l-alanine, alanine racemase of Bacillus subtilis in combination with d-amino acid oxidase of Rhodotorula gracilis or Trigonopsis variabilis respectively was introduced into the reaction system for the l-2-aminobutyric acid synthesis. l-Alanine could be completely removed by the action of alanine racemase of B. subtilis and d-amino acid oxidase of R. gracilis; thereby, high-purity l-2-aminobutyric acid was achieved. The results revealed that alanine racemase could discriminate effectively between l-alanine and l-2-aminobutyric acid, and selectively catalyzed l-alanine to d-alanine reversibly. d-Amino acid oxidase then catalyzed d-alanine to pyruvate stereoselectively. Furthermore, this method was also successfully used to remove the by-product l-alanine in the production of other neutral amino acids such as l-tertiary leucine and l-valine, suggesting that multienzymatic whole-cell catalysis can be employed to provide high purity products.     

Racemic Resolution of some <Emphasis Type="SmallCaps">dl</Emphasis>-Amino Acids using <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-Amino Acid Oxidase

The ability of Aspergillus fumigatus l-amino acid oxidase (l-aao) to cause the resolution of racemic mixtures of dl-amino acids was investigated with dl-alanine, dl-phenylalanine, dl-tyrosine, and dl-aspartic acid. A chiral column, Crownpak CR+ was used for the analysis of the amino acids. The enzyme was able to cause the resolution of the three dl-amino acids resulting in the production of optically pure d-alanine (100% resolution), d-phenylalanine (80.2%), and d-tyrosine (84.1%), respectively. The optically pure d-amino acids have many uses and thus can be exploited industrially. This is the first report of the use of A. fumigatus l-amino acid oxidase for racemic resolution of dl-amino acids.     

Cloning,Purification and Characterization of an NAD-Dependent <Emphasis Type="SmallCaps">d</Emphasis>-Arabitol Dehydrogenase from Acetic Acid Bacterium, <Emphasis Type="Italic">Acetobacter suboxydans</Emphasis>

d-Xylulose-forming d-arabitol dehydrogenase (aArDH) is a key enzyme in the bio-conversion of d-arabitol to xylitol. In this study, we cloned the NAD-dependent d-xylulose-forming d-arabitol dehydrogenase gene from an acetic acid bacterium, Acetobacter suboxydans sp. The enzyme was purified from A. suboxydans sp. and was heterogeneously expressed in Escherichia coli. The native or recombinant enzyme was preferred NAD(H) to NADP(H) as coenzyme. The active recombinant aArDH expressed in E. coli is a homodimer, whereas the native aArDH in A. suboxydans is a homotetramer. On SDS–PAGE, the recombinant and native aArDH give one protein band at the position corresponding to 28 kDa. The optimum pH of polyol oxidation and ketone reduction is found to be pH 8.5 and 5.5 respectively. The highest reaction rate is observed when d-arabitol is used as the substrate (K _m = 4.5 mM) and the product is determined to be d-xylulose by HPLC analysis.     

d-Aspartyl residue in a peptide can be liberated and metabolized by pig kidney enzymes

Summary The presence of an enzyme activity which hydrolyzes glycyl-d-aspartate was found in the homogenates of pig kidney cortex. The activity was inhibited by metal chelating agents and cilastatin, suggesting that the enzyme was a cilastatin-sensitive metallo-peptidase. Of the two hydrolysis products,d-aspartate was found to be less accumulated than glycine. The fate ofd-aspartate was, therefore, examined and the amino acid was found to be converted tol-aspartate,l-alanine and pyruvate, in the presence ofl-glutamate. Experiments with enzyme inhibitors suggested that the conversion involvedd-aspartate oxidase, aspartate aminotransferase and alanine aminotransferase as well as decarboxylation of oxaloacetate produced fromd-aspartate. All the results indicate that the enzymes in the pig kidney can liberate thed-aspartyl residue in the peptide and convert it to the compounds readily utilizable. The finding suggests a probable metabolic pathway of thed-aspartate-containing peptide.     

Cloning and expression of a tyrosinase from <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>: application in <Emphasis Type="SmallCaps">l</Emphasis>-DOPA biotransformation

Amplification of the tyrosinase gene (melO) from the genomic DNA of Aspergillus oryzae NCIM 1212 yielded a 1.6-kb product. This gene was cloned into pYLEX1, and the resulting pTyro-YLEX1 vector was transformed in Yarrowia lipolytica strain Po1g. A clone displaying the highest specific activity for tyrosinase (10.94 U/mg) was used for obtaining the complementary DNA (cDNA) and for protein expression studies. cDNA sequence analysis indicated the splicing of an intron present in the melO gene by Po1g. Native polyacrylamide gel electrophoresis, acidification at pH 3.0 followed by activity staining with l-DOPA indicated the expression of an active tyrosinase. The clone over-expressing the tyrosinase transformed l-tyrosine to l-DOPA. On optimization of conditions for the biotransformation (pH 4.0, temperature 60°C and with 3.5 mg of biomass), 0.4 mg/ml of l-DOPA was obtained.     

Increased <Emphasis Type="SmallCaps">d</Emphasis>-allose production by the R132E mutant of ribose-5-phosphate isomerase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Ribose-5-phosphate isomerase from Clostridium thermocellum converted d-psicose to d-allose, which may be useful as a pharmaceutical compound, with no by-product. The 12 active-site residues, which were obtained by molecular modeling on the basis of the solved three-dimensional structure of the enzyme, were substituted individually with Ala. Among the 12 Ala-substituted mutants, only the R132A mutant exhibited an increase in d-psicose isomerization activity. The R132E mutant showed the highest activity when the residue at position 132 was substituted with Ala, Gln, Ile, Lys, Glu, or Asp. The maximal activity of the wild-type and R132E mutant enzymes for d-psicose was observed at pH 7.5 and 80°C. The half-lives of the wild-type enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 11, 7.0, 4.2, 1.5, and 0.6 h, respectively, whereas those of the R132E mutant enzymes were 13, 8.2, 5.1, 3.1, and 0.9 h, respectively. The specific activity and catalytic efficiency (k _cat/K _m) of the R132E mutant for d-psicose were 1.4- and 1.5-fold higher than those of the wild-type enzyme, respectively. When the same amount of enzyme was used, the conversion yield of d-psicose to d-allose was 32% for the R132E mutant enzyme and 25% for the wild-type enzyme after 80 min.     

A gene encoding alanine racemase is involved in spore germination in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Alanine racemase is a major component of the exosporium of Bacillus cereus spores. A gene homologous to that of alanine racemase (alrA) was cloned from Bacillus thuringiensis subsp. kurstaki, and RT-PCR showed that alrA was transcribed only in the sporulating cells. Disruption of alrA did not affect the growth and sporulation of B. thuringiensis, but promoted l-alanine-induced spore germination. When the spore germination rate was measured by monitoring DPA release, complementation of the alrA disruptant reduced the rate of l-alanine-induced spore germination below that of even wild-type spores. As previously reported for spores of other Bacillus species, d-alanine was an effective and competitive inhibitor of l-alanine-induced germination of B. thuringiensis spores. d-cycloserine alone stimulated inosine-induced germination of B. thuringiensis spores in addition to increasing l-alanine-induced germination by inhibiting alanine racemase. d-Alanine also increased the rate of inosine-induced germination of wild-type spores. However, d-alanine inhibited inosine-induced germination of the alrA disruptant spores. It is possible that AlrA converted d-alanine to l-alanine, and this in turn, stimulated spore germination in B. thuringiensis. These results suggest that alrA plays a crucial role in moderating the germination rate of B. thuringiensis spores.     

Characterization of new <Emphasis Type="SmallCaps">l,d</Emphasis>-endopeptidase gene product CwlK (previous YcdD) that hydrolyzes peptidoglycan in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis has various cell wall hydrolases, however, the functions and hydrolase activities of some enzymes are still unknown. B. subtilis CwlK (YcdD) exhibits high sequence similarity with the peptidoglycan hydrolytic l,d-endopeptidase (PLY500) of Listeria monocytogenes phage and CwlK has the VanY motif which is a d-alanyl-d-alanine carboxypeptidase (Pfam: http://www.sanger.ac.uk/Software/Pfam/). The β-galactosidase activity observed on cwlK-lacZ fusion indicated that the cwlK gene was expressed during the vegetative growth phase, and Western blotting suggested that CwlK seems to be localized in the membrane. Truncated CwlK fused with a histidine-tag (h-ΔCwlK) was produced in Escherichia coli and purified on a nickel column. The h-ΔCwlK protein hydrolyzed the peptidoglycan of B. subtilis, and the optimal pH, temperature and NaCl concentration for h-ΔCwlK were pH 6.5, 37°C, and 0 M, respectively. Interestingly, h-ΔCwlK could hydrolyze the linkage of l-alanine-d-glutamic acid in the stem of the peptidoglycan, however, this enzyme could not hydrolyze the linkage of d-alanine-d-alanine, suggesting that CwlK is an l,d-endopeptidase not a d,d-carboxypeptidase. CwlK could not hydrolyze polyglutamate from B. natto or peptidoglycan of Staphylococcus aureus. This is the first report describing the characterization of an l,d-endopeptidase in B. subtilis and also the first report in bacteria of the characterization of a PLY500 family protein encoded in chromosomal DNA. Tatsuya Fukushima and Yang Yao contributed equally to this work.     

Zur chemischen Zusammensetzung der Zellwand der Streptokokken

Zusammenfassung Das Murein (Peptidoglycan) eines aus Faeces isolierten Streptococcus, der in den wichtigsten Merkmalen mit Peptostreptococcus evolutus (Prevot) Smith übereinstimmt, weist folgende Molverhältnisse auf (aufgerundete bzw. abgerundete Zahlen): Mur:GlcNH₂:Ala:Glu:Lys:Gly=1:1:3:1:1:1. Das Verhältnis l-Alanin:d-Alanin=2,15:1. Die Glutaminsäure liegt in der d-Konfiguration und als Amid vor.Durch die Partialhydrolyse der Zellwände und die anschließende Isolierung und Identifizierung der Peptide konnte die Aminosäuresequenz des Mureins geklärt werden. Das Tetrapeptid stimmt mit der üblichen Sequenz l-Ala-d-Glu-NH₂-l-Lys-d-Ala der meisten übrigen Bakterien überein. Die Quervernetzung des Mureins wird durch das Peptid Glycyl-l-Alanin hergestellt, wobei l-Alanin an die -Aminogruppe des Lysins gebunden ist. Die Dinitrophenylierung der Zellwand ergab, daß 35% des Glycins und 6% des Lysins eine freie Aminogruppe aufweisen. Die Quervernetzung ist demnach nur zu höchstens 60% durchgeführt.

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The chemical composition of the cell walls of Streptococci III. The amino acid sequence of a glycine containing murein from Peptostreptococcus evolutus (Prevot) Smith

Summary Peptostreptococcus evolutus was isolated from feces. Its murein containes muramic acid, glucosamine, alanine, d-glutamic acid, lysine and glycine at a molar ratio of about 1:1:3:1:1:1. The ratio of l-alanine: d-alanine is 2,15:1. Glutamic acid is present as an amide.By acid partial hydrolysis of the cell walls and subsequent isolation and identification of the peptides the amino acid sequence of the murein was elucidated. The tetrapeptide is identical with that of most bacteria (l-Ala-d-Glu-NH₂-l-Lys-d-Ala). The crosslinking of the murein is performed by the peptide glycyl-l-alanine. l-alanine is attached to the -amino group of lysine while the amino group of glycine is bound to the carboxyl group of the c-terminal d-alanine of an adjacent tetrapeptide. About 35% glycine and 6% lysine of the murein are dinitrophenylisable indicating that maximally 60% of the possible cross-linkages are realized.

     

Impact of Aspergillus oryzae genomics on industrial production of metabolites

Aspergillus oryzae is used extensively for the production of the traditional Japanese fermented foods sake (rice wine), shoyu (soy sauce), and miso (soybean paste). In recent years, recombinant DNA technology has been used to enhance industrial enzyme production by A. oryzae. Recently completed genomic studies using expressed sequence tag (EST) analyses and whole-genome sequencing are quickly expanding the industrial potential of the fungus in biotechnology. Genes that have been newly discovered through genome research can be used for the production of novel valuable enzymes and chemicals, and are important for designing new industrial processes. This article describes recent progress of A . oryzae genomics and its impact on industrial production of enzymes, metabolites, and bioprocesses.     

Die Wirkung von d-Aminosäuren auf die Struktur und Biosynthese des Peptidoglycans

Zusammenfassung In einem Konzentrationsbereich von 0,02–0,2 M hemmt d-Serin das Wachstum aller untersuchten Bakterien. Gleichzeitig traten morphologische Veränderungen der Bakterienzellen auf. In den nucleotidaktivierten Vorstufen von gehemmten Zellen wurden die d-Alaninreste des Peptidanteils ganz oder teilweise durch d-Serin ersetzt. Auch das Peptidoglycan enthielt d-Serin anstelle von d-Alanin, jedoch weiniger als in den Vorstufen. Zusätzlich war das modifizierte Peptidoglycan zu einem geringeren Anteil quervernetzt als das normale. Vier weitere d-Aminosäuren (Threonin, Valin, Leucin, Methionin) verursachten bei einer Konzentration von 0.2 M ähnliche Wirkungen wie d-Serin. Die Wirkungsweise von d-Aminosäuren auf die Peptidoglycansynthese kann daher allgemein wie folgt beschreiben werden: In Gegenwart von wachstumshemmenden Konzentrationen an d-Aminosäuren werden modifizierte nucleotidaktivierte Peptidoglycanvorstufen synthetisiert, die zu einem geringeren Ausmaß in das Peptidoglycan eingebaut und im Peptidoglycan schlechter quervernetzt werden. Der Ersatz von d-Alanin in Position 4 der Peptiduntereinheit ist dabei in der Regel am wirkungsvollsten. Nur bei Corynebacterium insidiosum und Staphylococcus aureus erwies sich der Ersatz in Position 5 als stärker hemmend. Diese Wirkungsweise entspricht weitgehend derjenigen von Glycin. Im Unterschied zur Wirkung von Glycin kann l-Alanin in Position 1 der Peptiduntereinheit nicht durch d-Aminosäuren ersetzt werden.

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Mode of action of d-amino acids on the biosynthesis of peptidoglycan

The mechanism of growth inhibition by d-amino acids was studied. d-Serine at concentrations from 0.02–0.2 M was sufficient to cause partial growth inhibition in seven species of bacteria representing the four most common types of peptidoglycan. The inhibited cells displayed morphological alterations. In the nucleotide-activated peptidoglycan precursors of these cells, d-alanine residues in position 4 and/or 5 of the peptide moiety were partially or even completely replaced by d-serine. The peptidoglycan also contained d-serine instead of d-alanine, but the percentual content of d-serine was significantly lower than that in the precursors. In addition, the modified peptidoglycan was less cross-linked than the normal one. Four other d-amino acids (d-threonine, d-valine, d-leucine, d-methionine) at concentrations of about 0.2 M caused similar effects as did d-serine when applied to Corynebacterium callunae and Bacillus subtilis. Thus the mode of action of d-amino acids on peptidoglycan synthesis can be generally described as follows: in their presence, at growth inhibiting concentrations modified nucleotide-activated peptidoglycan precursors are formed in which d-alanine residues are replaced by the d-amino acids. They are less efficiently incorporated into peptidoglycan. A high percentage of the modified muropeptides remains non-cross-linked, since they are poor substrates for the transpeptidation reaction. In the majority of the organisms, cross-linking was decreased when d-alanine in position 4 of the peptide subunit was replaced, in two organisms (Corynebacterium insidiosum and Staphylococcus aureus) replacement in position 5 was most effective, however. The low extent of crosslinkage is consistent with the morphological aberrations of inhibited cells. In previous studies with glycine, results were described that were in close analogy to those obtained with d-amino acids. However, glycine can replace not only d-alanine residues in position 4 and 5 but also l-alanine in position 1 of the peptide subunit.

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Verwendete Abkürzungen Dab Diaminobuttersäure - m-Dmp meso-Diaminopimelinsäure - GlcNAc oder G N-Acetylglucosamin - MurNAc oder M N-Acetylmuraminsäure     

Xanthomonas oryzae pv. oryzae infection triggers accumulation of phenolics,defense-related enzymes and thaumatin-like proteins in rice leaves

Abstract

The effect of Xanthomonas oryzae pv. oryzae infection on induction of phenylalanine ammonia-lyase (PAL), peroxidase (PO), phenolics and thaumatin-like proteins (TLPs) in rice was studied. PAL activity increased significantly one day after inoculation with X. o. pv. oryzae and the maximum enzyme activity was observed two days after inoculation. The phenolic content in rice leaves increased significantly one day after inoculation and the maximum accumulation of phenols was observed two days after inoculation. Significant increase in peroxidase activity was observed in rice leaves one day after inoculation with X. o. pv. oryzae. Isozyme analysis indicated that three peroxidase isozymes (PO-1, PO-2 and PO-3) were induced after inoculation with X. o. pv. oryzae. Immunoblot analysis of protein extracts from control and pathogen inoculated rice plants revealed the induced accumulation of 16 and 24 kDa TLPs in rice leaves in response to X. o. pv. oryzae infection. TLP mRNA accumulation was induced strongly in rice leaves in response to infection by X. o. pv. oryzae.     

Utilization of d-amino acids by dadR mutants of Salmonella typhimurium

Utilization of d-amino acids being substrates of d-amino acid dehydrogenase of Salmonella typhimurium was examined. The experiments were done with wild type strains and the mutants dadA missing the enzyme activity and dadR in which its synthesis is released from catabolite repression. Growth on d-tryptophan, d-histidine and d-methionine used as precursors of the l-amino acids was faster when the respective auxotrophs carried dadR mutations. The dadR mutants grew faster when d-or l-alanine was present as a sole source of nitrogen. Experiments with d-amino acid dehydrogenase in vitro provided evidence that d-tryptophan is its substrate with a very low affinity to the dehydrogenase.     

Inhibition ofd-alanyl-d-alanine ligase in different bacterial species by amino phosphonic acids

A comparative study was performed on the kinetic properties and the specificity ofd-alanyl-d-alanine ligases fromPseudomonas aeruginosa, Streptococcus faecalis, andStaphylococcus aureus, using some aminophosphonic acids and related compounds.dl-I-Aminoethylphosphonic acid was shown to be a competitive inhibitor of theP. aeruginosa andS. faecalis ligases; assuming ad-form stereospecificity, its activity was nearly equal to that ofd-cycloserine. 2-Aminoethylphosphonate was found to be a weak inhibitor of the ligases, in contrast to the carboxylic analog, β-alanine. γ-Aminobutyric acid and phosphoethanolamine also exhibited some inhibitory properties.