Insensitivity of D-amino acid dehydrogenase synthesis to catabolic repression in dadR mutants of Salmonella typhimurium

It has been found that synthesis of D-amino acid dehydrogenase in Salmonella typhimurium is stimulated by cyclic AMP and crp gene product. This indicates that catabolic control of the dehydrogenase resembles other bacterial systems of catabolic repression. We have isolated S. typhimurium mutants, dadR, which are resistant to L-methionine-interference with D-histidine utilization and are able to utilize D-tryptophan as a precursor of L-tryptophan. Mapping data indicate that the dadR locus is closely linked to dadA coding for the structure of D-amino acid dehydrogenase. The synthesis of the dehydrogenase in dadR mutants is completely insensitive to the repression by glucose, but remains inducible by L-alanine. We conclude thereof that dadR mutants have changes in the promoter region which increase the expression of the dadA gene in the presence of glucose metabolism. A likely possibility that induction of the dad operon by alanine might be under positive control is discussed.     

Role of D-tryptophan oxidase in D-tryptophan utilization by Escherichia coli.

Mutants of Escherichia coli K-12 that require L-tryptophan (trp) are normally unable to utilize D-tryptophan to fulfill their requirement. However, secondary mutations (dadR) that confer this ability can be isolated. In such strains two distinct enzymes are found to be produced at high levels: D-amino acid oxidase (EC 1.4.3.3) and D-tryptophan oxidase. A convenient assay procedure for D-tryptophan oxidase is described. The two enzymes could be distinguished on the basis of their sensitivity to inhibition by L-phenylalanine and L-tyrosine. Strains that were trp dadR could not grow with D-tryptophan in the presence of L-phenylalanine, but further mutations, Fyo, could be isolated that allowed growth under these conditions. Some of them were characterized by further increases in the level of D-tryptophan oxidase activity and a sharp decrease in D-amino acid oxidase. These kinds of Fyo mutations lay in or near the dadR gene. The substrate specificity of the two enzymes toward a large number of compounds was examined. The transamination of aromatic keto acids was investigated. In the wild-type strain only a single enzyme, transaminase A (EC 2.6.1.5), was found, and it was irreversibly activated when subjected to elevated temperatures. The present state of our knowledge on D-amino acid utilization in E. coli is summarized.     

Evolutionary significance of the system utilizing D-amino acid enantiomers.

The evolutionary significance of the system utilizing D-amino acid enantiomers in living organisms is discussed, based on an experiment in which the mutant from Escherichia coli K--12 4627 grown on D-tryptophan was used. The mutant shows the ability of D-tryptophan is degraded to indol which can be utilized for the synthesis of L-tryptophan in the presence of serine.     

Induction of N–malonyl–D–tryptophan by drought stress. Is D–tryptophan the only D–amino acid appeared in wilted leaves?

D-amino acid were searched in wilted tomato leaves. D-Isomers of free amino acids were not revealed by the treatment with L- and D-amino acid oxidases. The noncationic fraction of the extract contained N-malonyl-D-tryptophan and no other N-acylated amino acids. A special search for endogenous N-malonyl-D-phenylalanine gave negative results. Exogenous¹⁴C-malonate was only incorporated in one Chromatographic zone corresponding to N-malonyl-D-tryptophan. It is concluded that drought stress does not induce the appearance of D-amino acids except for D-tryptophan which is accumulated in the malonylated form.     

A new D-stereospecific amino acid amidase from Ochrobactrum anthropi

A new D-stereospecific amino acid amidase has been partially purified from Ochrobactrum anthropi SCRC SV3, which had been isolated and selected from soil. The Mr of the enzyme was estimated to be about 38,000, and its isoelectric point was 5.3. The enzyme catalyzes the stereospecific hydrolysis of D-amino acid amide to yield D-amino acid and ammonia. The major substrates included D-phenylalanine amide, D-tyrosine amide, D-tryptophan amide, D-leucine amide, and D-alanine amide.     

Formation of D-tyrosyl-tRNATyr accounts for the toxicity of D-tyrosine toward Escherichia coli

D-Tyr-tRNATyr deacylase cleaves the ester bond between a tRNA molecule and a D-amino acid. In Escherichia coli, inactivation of the gene (dtd) encoding this deacylase increases the toxicity of several D-amino acids including D-tyrosine, D-tryptophan, and D-aspartic acid. Here, we demonstrate that, in a Deltadtd cell grown in the presence of 2.4 mm D-tyrosine, approximately 40% of the total tRNATyr pool is converted into D-Tyr-tRNATyr. No D-Tyr-tRNATyr is observed in dtd+ cells. In addition, we observe that overproduction of tRNATyr, tRNATrp, or tRNAAsp protects a Deltadtd mutant strain against the toxic effect of D-tyrosine, D-tryptophan, or D-aspartic acid, respectively. In the case of D-tyrosine, we show that the protection is accounted for by an increase in the concentration of L-Tyr-tRNATyr proportional to that of overproduced tRNATyr. Altogether, these results indicate that, by accumulating in vivo, high amounts of D-Tyr-tRNATyr cause a starvation for L-Tyr-tRNATyr. The deacylase prevents the starvation by hydrolyzing D-Tyr-tRNATyr. Overproduction of tRNATyr also relieves the starvation by increasing the amount of cellular L-Tyr-tRNATyr available for translation.     

Enantioselective synthesis of various D-amino acids by a multi-enzyme system

We have developed an effective method for the synthesis of various D-amino acids from the corresponding α-keto acids and ammonia by coupling four enzyme reactions catalyzed by D-amino acid aminotransferase, glutamate racemase, glutamate dehydrogenase, and formate dehydrogenase. In this system, D-glutamate is continuously regenerated from α-ketoglutarate, ammonia and NADH by the coupled reaction of glutamate dehydrogenase and glutamate racemase, and used as an amino donor for the enantioselective D-amino acid synthesis by the D-amino acid aminotransferase reaction. The unidirectional formate dehydrogenase reaction is also coupled to regenerate NADH consumed. Under the optimum conditions, D-enantiomers of valine, alanine, α-keto analogues with a molar yield higher than 80%.     

D-amino acid dehydrogenase: the enzyme of the first step of D-histidine and D-methionine racemization in Salmonella typhimurium

Summary Mutants ofSalmonella typhimurium deficient in D-amino acid dehydrogenase were isolated in histidine auxotrophs able to utilize D-histidine(his-dhuA) 1. The mutants have lost the ability to utilize D-histidine and D-methionine due to mutations in the locusdadA mapped in co-transducible vicinity of the genehemA. ThedadA mutants were unable to deaminate D-histidine, D-methionine, D-alanine and several other D-amino acids to the respective keto products. Indad + strains the enzyme activity was the highest in toluenized cells. In crude sonieates it was 5 to 10 times less. Reduction of artificial electron accepters in the presence of D-amino acids behaved similarly. Keto product formation was strongly inhi-bited by cyanide. It has been concluded thereof that the deaminating enzyme is a D-amino acid dehydrogenase, the activity of which depends on structural integrity of a cell component or on a structure-bound electron accepter. The enzyme activity was inducible by adding L-or D-alanine to growth media. The induction was the highest in media with poor carbon sources. A temperature-sensitivedadA mutant was isolated. I t mapped indadA and had thermolabile D-amino acid dehydrogenase. This has indicated thatdadA is structural gene for the D-amino acid dehydrogenase. This work was supported by the Polish Academy of Sciences within the project 09.3.1., and by the U.S. Public Health Service, grant No. 05-032-1. The nomenclature rules for describing genotypes and phenotypes of Demerecet al. (1966) were followed throughout this paper. E.g.dhuA ⁻ hisP⁺ mutants have Dhu⁺ phenotype, those with dhuA^{s - hisP s - mutations are phenotypically Dhu}- All strains with wild-type dhuA⁺ lOCUS are Dhu⁻.     

Efficiency of D-tryptophan as Niacin in rats

L-tryptophan is a very important precursor of niacin in mammals. Food preparation in which proteins are exposed to an alkali and/or high temperature for a long period generate appreciable amounts of D-amino acids from racemization. The efficiency of D-tryptophan as niacin was thus investigated by using weanling rats. The availability of D-tryptophan was almost the same as that in L-tryptophan as the precursor of niacin and was 1/6 as active as niacin.     

Purification, characterization, gene cloning and nucleotide sequencing of D-stereospecific amino acid amidase from soil bacterium: Delftia acidovorans

The D-amino acid amidase-producing bacterium was isolated from soil samples using an enrichment culture technique in medium broth containing D-phenylalanine amide as a sole source of nitrogen. The strain exhibiting the strongest activity was identified as Delftia acidovorans strain 16. This strain produced intracellular D-amino acid amidase constitutively. The enzyme was purified about 380-fold to homogeneity and its molecular mass was estimated to be about 50 kDa, on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active preferentially toward D-amino acid amides rather than their L-counterparts. It exhibited strong amino acid amidase activity toward aromatic amino acid amides including D-phenylalanine amide, D-tryptophan amide and D-tyrosine amide, yet it was not specifically active toward low-molecular-weight D-amino acid amides such as D-alanine amide, L-alanine amide and L-serine amide. Moreover, it was not specifically active toward oligopeptides. The enzyme showed maximum activity at 40 degrees C and pH 8.5 and appeared to be very stable, with 92.5% remaining activity after the reaction was performed at 45 degrees C for 30 min. However, it was mostly inactivated in the presence of phenylmethanesulfonyl fluoride or Cd2+, Ag+, Zn2+, Hg2+ and As3+ . The NH2 terminal and internal amino acid sequences of the enzyme were determined; and the gene was cloned and sequenced. The enzyme gene damA encodes a 466-amino-acid protein (molecular mass 49,860.46 Da); and the deduced amino acid sequence exhibits homology to the D-amino acid amidase from Variovorax paradoxus (67.9% identity), the amidotransferase A subunit from Burkholderia fungorum (50% identity) and other enantioselective amidases.     

Evidence for the presence of two D-amino acid oxidases inPseudomonas aeruginosa PAO

By the isolation of mutants that were unable to grow on L-hydroxyproline or DL-valine, it has been possible to demonstrate the presence of two different types of D-amino acid oxidase activities inPseudomonas aeruginosa PAO. One was the D-amino acid dehydrogenase, probably involved in the oxidation of a number of D-amino acids such as D-alanine, D-phenylalanine and D-valine. The other was the inducible oxidase, specific to the oxidation of allohydroxy-D-proline formed from L-hydroxyproline during its oxidation. Thus, it has been possible to delink the involvement of the general D-amino acid dehydrogenase in the oxidative breakdown of allohydroxy-Dsproline.     

Immobilization and characterization of D-amino acid oxidase.

Optimal conditions with respect to pH, concentration of glutaraldehyde and enzyme, and order of addition of enzyme and crosslinking reagent were established for the immobilization of hog kidney D-amino acid oxidase to an attapulgite support. Yields of 40 to 70% were generally attained although when low concentrations of enzyme were used yields were consistently greater than 100%. It is suggested that this is due to a dimer leads to monomer shift at low protein concentrations. The stability of soluble D-amino acid oxidase was dependent on the buffer in which it was stored (pyrophosphate-phosphate greater than borate greater than Tris). Stability of immobilized enzyme was less than soluble in pyrophosphate-phosphate buffer, but storage in the presence of FAD improved stability. In addition, treatment of stored, immobilized enzyme with FAD before assay restored some of its activity. The immobilized D-amino acid oxidase was less stable to heat (50 degrees C) than the soluble enzyme from pH 6 to 8 but was more stable above and below these values. Apparent Km values for D-alanine, D-valine, and D-tryptophan decreased for the immobilized enzyme compared to the soluble.     

荧光假单胞菌TM5-2中D-型氨基酸脱氢酶的鉴定和表达

从荧光假单胞菌TM5-2中得到一个含丙氨酸消旋酶基因的DNA片段(8.8kb),相邻的一个开读框(ORF)与甘氨酸/D-型氨基酸氧化酶基因相似。该ORF经过克隆、表达,并没有检测到甘氨酸/D-型氨基酸氧化酶的活性,推导而得的氨基酸序列与D-型氨基酸脱氢酶序列比较发现,ORF含有D-型氨基酸脱氢酶的所有重要的保守序列。经TTC培养基鉴定,其具有D-型氨基酸脱氢酶的活性,并对一系列D-型氨基酸有作用,最佳作用底物是D-组氨酸。     

Isolation of peroxisomes from the dog kidney cortex.

The present study was undertaken to separate peroxisomes of the dog kidney cortex by the methods of discontinuous sucrose density gradient and zonal centrifugation. The separation of subcellular particles was evaluated by measuring the activities of reference enzymes, beta-glycerophosphatase for lysosomes, succinate dehydrogenase for mitochondria, glucose-6-phosphatase for microsomes, and catalase and D-amino acid oxidase for peroxisomes. The activities of D-amino acid oxidase and catalase were mainly observed in fractions 1 and 2 (1.6 and 1.7 M sucrose) obtained by discontinuous sucrose density-gradient centrifugation. Small amounts of acid phosphatase and succinate dehydrogenase contaminated these fractions. Considerably higher activity of catalase was determined in the supernatant, while D-amino acid oxidase showed a lower activity. By the method of zonal centrifugation, the highest specific activities of catalase and D-amino acid oxidase were found in fraction 50 (1.73 M sucrose) with no succinate dehydrogenase, acid phosphatase or glucose-6-phosphatase activity. These results suggested that peroxisomes of dog kidney cortex were clearly separated in 1.73 M sucrose from mitochondria, lysosomes and microsomes by zonal centrifugation.     

Esterification of Escherichia coli tRNAs with D-histidine and D-lysine by aminoacyl-tRNA synthetases

It is generally believed that only L-amino acids are acceptable in protein synthesis, though some D-amino acids, including D-tyrosine, D-aspartate, and D-tryptophan are known to be bound enzymatically to tRNAs. In this report, we newly show that D-histidine and D-lysine are also able to be the substrates of respective Escherichia coli aminoacyl-tRNA synthetases.     

Enzymologic study of the structural organization of the matrix or rat liver peroxisomes]

The effect of ionic strength and pH on the release of some enzymes of the matrix of peroxisomes in rat's liver was studied. Catalase, L ALpha-hydroxy acid oxidase, isocitrate dehydrogenase, glycerophosphate dehydrogenase and lactate dehydrogenase were easily released from the particles during their lysis and treatment with 0.16 M KCl, whereas urate oxidase, NADH cytochrome c reductase and D-amino acid oxidase were not solubilized. After the solubilization of peroxisomal membrane by 0.2% Triton X-100, the remaining core contained about 50% amino acid oxidase activity, and had 1.28--1.30 g/cm3 density. These results suggest that D-amino acid oxidase associates with urate oxidase in the peroxisomal core.     

Some N-acyl-D-amino acid derivatives having antibotulinal properties

Several N-acyl derivatives of D-tryptophan, D-alanine, D-methionine, D-valine, and D-aspartic acid were synthesized in high yields using the succinimidyl ester method and examined for their antibotulinal properties. In conjunction with 60 ppm of sodium nitrite, sorbyl-D-tryptophan, sorbyl-D-alanine, myristoyl-D-aspartic acid, and glycyl-D-alanine were highly inhibitory. In the absence of sodium nitrite, the N-acyl derivatives of the D-amino acids were not inhibitory. On its own, 60 ppm of sodium nitrite was only slightly inhibitory. Sorbyl-L-tryptophan and sorbyl-L-alanine had no effect in the presence or absence of 60 ppm of sodium nitrite.     

内消旋-二氨基庚二酸脱氢酶不对称合成非天然手性D-氨基酸的研究进展

内消旋-二氨基庚二酸脱氢酶不对称合成非天然的手性D-氨基酸是目前生物催化领域的研究热点。内消旋-二氨基庚二酸脱氢酶具有优良的立体选择性,利用其进行酶催化不对称合成光学纯的手性D-氨基酸,被广泛用于医药、食品、化妆品、精细化学品等领域。为了促进生物催化法在合成手性D-氨基酸方向的进一步发展,本文对内消旋-二氨基庚二酸脱氢酶催化合成D-氨基酸的现状进行了综述。重点介绍了Corynebacterium glutamicum、Ureibacillus thermosphaericus、Symbiobacterium thermophilum来源的内消旋-二氨基庚二酸脱氢酶在新酶的挖掘、催化性能、晶体结构解析、分子改造、功能与催化机制、合成D-氨基酸新途径等方面的研究进展,并对内消旋-二氨基庚二酸脱氢酶的未来研究方向及策略进行了展望。本综述将进一步加深人们对内消旋-二氨基庚二酸脱氢酶的认识,也为具有挑战性的生物合成任务提供信息借鉴。     

The three-dimensional structure of the ternary complex of Corynebacterium glutamicum diaminopimelate dehydrogenase-NADPH-L-2-amino-6-methylene-pimelate

The three-dimensional (3D) structure of Corynebacterium glutamicum diaminopimelate D-dehydrogenase in a ternary complex with NADPH and L-2-amino-6-methylene-pimelate has been solved and refined to a resolution of 2.1 A. L-2-Amino-6-methylene-pimelate was recently synthesized and shown to be a potent competitive inhibitor (5 microM) vs. meso-diaminopimelate of the Bacillus sphaericus dehydrogenase (Sutherland et al., 1999). Diaminopimelate dehydrogenase catalyzes the reversible NADP+ -dependent oxidation of the D-amino acid stereocenter of mesodiaminopimelate, and is the only enzyme known to catalyze the oxidative deamination of a D-amino acid. The enzyme is involved in the biosynthesis of meso-diaminopimelate and L-lysine from L-aspartate, a biosynthetic pathway of considerable interest because it is essential for growth of certain bacteria. The dehydrogenase is found in a limited number of species of bacteria, as opposed to the alternative succinylase and acetylase pathways that are widely distributed in bacteria and plants. The structure of the ternary complex reported here provides a structural rationale for the nature and potency of the inhibition exhibited by the unsaturated L-2-amino-6-methylene-pimelate against the dehydrogenase. In particular, we compare the present structure with other structures containing either bound substrate, meso-diaminopimelate, or a conformationally restricted isoxazoline inhibitor. We have identified a significant interaction between the alpha-L-amino group of the unsaturated inhibitor and the indole ring of Trp144 that may account for the tight binding of this inhibitor.     

Solubilisation of D-amino acid dehydrogenase of Escherichia coli K12 and its re-binding to envelope preparations

D-amino acid dehydrogenase was found to be solubilised from envelope preparations of Escherichia coli by treatment with detergents but not with aqueous buffer solutions. Triton X-100-solubilised dehydrogenase was found to rebind to envelope preparations from the wild strain (AB 259) and its D-amino acid dehydrogenase-less mutant (DAD 13), and activities higher than those of native envelopes could be obtained. Re-binding was stimulated by magnesium. D-alanine stimulated cytochrome reduction and oxygen uptake were reconstituted when the solubilised dehydrogenase rebound to AB 259 envelopes. Re-binding of solubilised dehydrogenase to DAD 13 envelopes was independent of the growth medium used for DAD 13.