Allohydroxy-d-proline dehydrogenase

Growth of Pseudomonas aeruginosa PA01 on isomers of hydroxyproline induced the synthesis of an allohydroxy-d-proline dehydrogenase. The enzyme resembled the d-alanine dehydrogenase of this organism in its association with the particulate fraction and its linkage to oxygen through a cytochrome-containing respiratory chain, but differed from this and other bacterial d-amino acid dehydrogenases in its high substrate specificity and low K _m .     

d-Alanine metabolism in the lucinid clam Lucinoma aequizonata

The chemoautotrophic symbiont-bearing clam Lucinoma aequizonata contains very high levels of free d-alanine in all tissues. The possible sources for this amino acid and its involvement in the clams' metabolism were investigated. Very low levels of d-alanine (generally below 1 mol·l^-1) were measured in the sediment porewaters from the habitat of the clams. Experiments with ¹⁴C-labeled tracers demonstrate an active metabolism of d-alanine in the clams rather than a role as inert waste product. d-alanine is metabolized at about 0.12 mol·g fw^-1·h^-1. Label from aspartate, but not glucose and CO₂, is incorporated into d-alanine. Incubation with labeled d-alanine did not result in formation of radioactive l-alanine. Tests for alanine racemase (EC 5.1.1.1) and d-amino acid oxidase (EC 1.4.3.3.) did not show activity in either gill, i.e. symbiont and host, or foot tissue. d-Alanine amino transferase (EC 2.6.1.b.) was demonstrated in gill and foot tissues. Two sources for d-alanine are proposed: a degradation of cell walls of symbiotic bacteria and production by the host using a d-specific alanine transaminase.Abbreviations aa amino acid(s) - fw fresh weight - HPLC high-performance liquid chromatography - MBH methyl benzethonium hydroxyde - NAC N-acetyl-l-cysteine - OPA ortho-phthaldialdehyde - TCA tricarbonic acid     

An expedient synthesis of l-ribulose and derivatives

A significant improvement in the production of l-ribulose from inexpensive and commercially available starting materials, l-arabinose and sodium aluminate, is demonstrated. This has facilitated expeditious access to gram-scale quantities of l-ribulofuranoside derivatives.     

d-Mannitol dehydrogenase and d-mannitol-1-phosphate dehydrogenase in Platymonas subcordiformis: some characteristics and their role in osmotic adaptation

d-Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and d-mannitol dehydrogenase (EC 1.1.1.67) were estimated in a cell-free extract of the unicellular alga Platymonas subcordiformis Hazen (Prasinophyceae), d-Mannitol dehydrogenase had two activity maxima at pH 7.0 and 9.5, and a substrate specifity for d-fructose and NADH or for d-mannitol and NAD⁺. The K _m values were 43 mM for d-fructose and 10 mM for d-mannitol. d-Mannitol-1-phosphate dehydrogenase had a maximum activity at pH 7.5 and was specific for d-fructose 6-phosphate and NADH. The K _m value for d-fructose 6-phosphate was 5.5 mM. The reverse reaction with d-mannitol 1-phosphate as substrate could not be detected in the extract. After the addition of NaCl (up to 800 mM) to the enzyme assay, the activity of d-mannitol dehydrogenase was strongly inhibited while the activity of d-mannitol-1-phosphate dehydrogenase was enhanced. Under salt stress the K _m values of the d-mannitol dehydrogenase were shifted to higher values. The K _m value for d-fructose 6-phosphate as substrate for d-mannitol-1-phosphate dehydrogenase remained constant. Hence, it is concluded that in Platymonas the d-mannitol pool is derectly regulated via alternative pathways with different activities dependent on the osmotic pressure.Abbreviations Fru6P d-fructose 6-phosphate - Mes 2-(N-morpholino)ethanesulfonic acid - MT-DH d-mannitol-dehydrogenase - MT1P-DH d-mannitol-1-phosphate dehydrogenase - Pipes 1,4-piperazinediethanesulfonic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

N-acetyl-l-glutamate in brain: Assay,levels, and regional and subcellular distribution

N-Acetyl-L-glutamate (NAG), the activator of mitochondrial carbamoyl phosphate synthetase (CPS), is demonstrated by several methods, including a new HPLC assay, in the brain of mammals and of chicken. The brain levels of NAG are 200–300 times lower than the levels of N-acetyl-l-aspartate (NAA), and are similar to the levels of NAG in rat liver. The NAG levels in chicken liver are very low. Although NAG is mitochondrial in the liver, it is cytosolic in brain. Using enzyme activity and immuno assays we did not detect CPS in brain (detection limit, 12.5 g/g brain), excluding that brain NAG is involved in citrullinogenesis. The regional distribution of brain NAG differs from that of NAA and resembles that of N-acetyl-l-aspartyl-l-glutamate (NAAG), suggesting that NAG and NAAG are related. NAG might be involved in the modulation of NAAG degradation.Special issue dedicated to Dr. Santiago Grisolía     

Dietary protein reduction in sheep and goats: different effects on <Emphasis Type="SmallCaps">l</Emphasis>-alanine and <Emphasis Type="SmallCaps">l</Emphasis>-leucine transport across the brush-border membrane of jejunal enterocytes

It was the aim of this study to examine the potential regulatory effects of a long-term low dietary protein supply on the transport capacity of the jejunal brush-border membrane for amino acids. For this purpose, we used the neutral amino acids L-alanine (representative for nonessential amino acids) and L-leucine (representative for essential amino acids) as model substances. Ten sheep lambs, 8 weeks of age and 19-27 kg body weight, were allotted to two dietary regimes with either adequate or reduced protein supply which was achieved by 17.9% and 9.7% of crude protein in the concentrated feed, respectively. The feeding periods were 4-6 weeks in length. Similarly, eight goat kids of 5-7 weeks of age and 8-14 kg body weight were allotted to either adequate (crude protein 20.1%, feeding period 9-12 weeks) or reduced protein supply (10.1%, feeding period 17-18 weeks). Dietary protein reduction in lambs caused a significant body weight loss of 0.6 +/- 0.7 kg, whereas the body weight in control animals increased by 1.9 +/- 0.7 kg (P<0.05). Plasma urea concentrations decreased significantly by 60% (low protein 2.3 +/- 0.1 versus control 5.7 +/- 0.2 mmol l(-1), P<0.001). In kids, reduction of dietary protein intake led to significant decreases of the daily weight gain by 48% from 181 +/- 8 g to 94 +/- 3 g (P<0.001) and daily dry matter intake by 27% from 568 +/- 13 g to 417 +/- 6 g (P<0.01). Respective urea concentrations in plasma were reduced by 77% from 5.2 +/- 0.4 to 1.2 +/- 0.2 mmol l(-1) (P<0.01). Kinetic analyses of the initial rates of alanine uptake into isolated jejunal brush-border membrane vesicles from sheep and goats as affected by low dietary protein supply yielded that the apparent Km was neither significantly different between the species nor significantly affected by the feeding regime thus ranging between 0.12 and 0.16 mmol.l(-1). Reduction of dietary protein, however, resulted in significantly decreased Vmax values of the transport system by 25-30%, irrespective of the species. Kinetic analyses of the initial rates of leucine uptake into jejunal brush-border membrane vesicles from sheep and goats yielded that leucine uptake was mediated by Na+-dependent as well as Na+-independent processes. Similar to alanine, apparent Km values of leucine uptake were neither different between the species nor affected due to low dietary protein and ranged between 0.08 and 0.15 mmol l(-1). In contrast to the alanine transport mechanism, dietary protein reduction resulted in increased Vmax values of Na+-dependent leucine transport by 53% in sheep and 230% in goats. Similarly, Na+-independent leucine uptake was stimulated by 85% and 200% in sheep and in goats, respectively. This study shows adaptation of amino acid absorption at the brush-border membrane level of jejunal enterocytes of small ruminants due to dietary protein reduction. Whereas the transport capacity for the nonessential amino acid alanine was reduced due to low dietary protein, the transport capacity for the essential amino acid leucine was markedly stimulated. From this, the involvement of rather different feedback mechanisms in adaptation of intestinal amino acid transport mechanisms has to be discussed.     

Importance of the l-galactonolactone pool for enhancing the ascorbate content revealed by l -galactonolactone dehydrogenase-overexpressing tobacco plants

l-Galactono-1,4-lactone (GalL) dehydrogenase (GLDH) is an enzyme that catalyzes the last step of l-ascorbate (AsA) biosynthesis in plants. To re-evaluate the importance of the enzyme and the possibility of manipulating the AsA content in plants, a cDNA encoding GLDH from sweet potato was introduced into tobacco plants by Agrobacterium-mediated transformation under the control of a CaMV 35S promoter. Protein blot analysis revealed the elevation of GLDH protein contents in three GLDH-transformed lines. Furthermore, these transgenic lines showed 6- to 10-fold higher GLDH activities in the roots than the non-transformed plants, SR1. Despite the elevated GLDH activity, the AsA content in the leaves did not change in all lines; i.e., the AsA content in GLDH-transformed lines was 3–7 μmol g⁻¹ FW, comparable to that in the non-transformed plants. Incubation of leaf discs in a GalL solution led to a rapid 2- to 3-fold increase in the AsA content in both GLDH-transformed and non-transformed plants in the same manner. These results suggest that the supply of GalL is a crucial factor for determining the AsA pool size and that the upstream genes in the AsA biosynthetic pathway are responsible for enhancing the AsA content in plants.     

Visible wavelength spectrophotometric assays of l-aspartate and d-aspartate using hyperthermophilic enzyme systems

Methods with which to simply and rapidly assay l-aspartate (l-Asp) and d-aspartate (d-Asp) would be highly useful for physiological research and for nutritional and clinical analyses. Levels of l- and d-Asp in food and cell extracts are currently determined using high-performance liquid chromatography. However, this method is time-consuming and expensive. Here we describe a simple and specific method for using an l-aspartate dehydrogenase (l-AspDH) system to colorimetrically assay l-Asp and a system of three hyperthermophilic enzymes—aspartate racemase (AspR), l-AspDH, and l-aspartate oxidase (l-AO)—to assay d-Asp. In the former, the reaction rate of nicotinamide adenine dinucleotide (NAD⁺)-dependent l-AspDH was measured based on increases in the absorbance at 438 nm, reflecting formation of formazan from water-soluble tetrazolium-1 (WST-1), using 1-methoxy-5-methylphenazinum methyl sulfate (mPMS) as a redox mediator. In the latter, d-Asp was measured after first removing l-Asp in the sample solution with l-AO. The remaining d-Asp was then changed to l-Asp using racemase, and the newly formed l-Asp was assayed calorimetrically using NAD⁺-dependent aspartate dehydrogenase as described above. This method enables simple and rapid spectrophotometric determination of 1 to 100 μM l- and d-Asp in the assay systems. In addition, methods were applicable to the l- and d-Asp determinations in some living cells and foods.     

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.     

The alternative<Emphasis Type="SmallCaps"> d</Emphasis>-galactose degrading pathway of<Emphasis Type="Italic"> Aspergillus nidulans</Emphasis> proceeds via<Emphasis Type="SmallCaps"> l</Emphasis>-sorbose

The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway. In contrast, Aspergillus nidulans mutants in galactokinase (galE) can still grow on d-galactose in the presence of ammonium—but not nitrate—ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme (araA1) did not show NAD⁺-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase (frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD⁺-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose.     

Origin ofd-serine present in urine of mutant mice lackingd-amino-acid oxidase activity

Summary Urine of ddY/DAO mice lackingd-amino-acid oxidase contained 5.7 times more serine than that of normal ddY/DAO⁺ mice. Most of the serine wasd-isomer. The origin of this^d-serine was examined. Oral administration of 0.02% amoxicillin and 0.004% minocycline to the ddY/ DAO^- mice for 7 days did not reduce the urinaryd-serine, indicating that thed-serine was not of intestinal bacterial origin. When the mouse diet was changed to one with different compositions, the urinaryd-serine was considerably reduced. Furthermore, starvation of the ddY/DAO^- mice for 24 hours reduced the urinaryd-serine to 33% of the original level. These results indicate that most of the urinaryd-serine comes from the diet. However, the urine of the starved ddY/DAO^- mice still contained 4.6 times mored-serine than that of the ddY/DAO⁺ mice, suggesting a part of the D-serine have an endogenous origin.     

Effect of pyruvate dehydrogenase complex deficiency on <Emphasis Type="SmallCaps">l</Emphasis>-lysine production with <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Intracellular precursor supply is a critical factor for amino acid productivity of Corynebacterium glutamicum. To test for the effect of improved pyruvate availability on l-lysine production, we deleted the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex (PDHC) in the l-lysine-producer C. glutamicum DM1729 and characterised the resulting strain DM1729-BB1 for growth and l-lysine production. Compared to the host strain, C. glutamicum DM1729-BB1 showed no PDHC activity, was acetate auxotrophic and, after complete consumption of the available carbon sources glucose and acetate, showed a more than 50% lower substrate-specific biomass yield (0.14 vs 0.33 mol C/mol C), an about fourfold higher biomass-specific l-lysine yield (5.27 vs 1.23 mmol/g cell dry weight) and a more than 40% higher substrate-specific l-lysine yield (0.13 vs 0.09 mol C/mol C). Overexpression of the pyruvate carboxylase or diaminopimelate dehydrogenase genes in C. glutamicum DM1729-BB1 resulted in a further increase in the biomass-specific l-lysine yield by 6 and 56%, respectively. In addition to l-lysine, significant amounts of pyruvate, l-alanine and l-valine were produced by C. glutamicum DM1729-BB1 and its derivatives, suggesting a surplus of precursor availability and a further potential to improve l-lysine production by engineering the l-lysine biosynthetic pathway. This study is dedicated to Prof. Dr. Hermann Sahm on the occasion of his 65th birthday.     

Microbial metabolism of d- and l-phenylglycine by Pseudomonas putida LW-4

A strain of Pseudomonas putida capable of utilizing both stereoisomers of phenylglycine as the sole carbon and energy source was isolated from soil. No phenylglycine racemase was detected in cells grown on either stereoisomer. In an initial reaction each steroisomer of phenylglycine was transaminated yielding phenylglyoxylate which was further metabolized via benzaldehyde to benzoate. Subsequently, benzoate was further degraded via an ortho-cleavage of catechol.Abbreviation HPLC high-performance liquid chromatography     

Structural studies of the O-specific polysaccharide from the lipopolysaccharide of Mesorhizobium huakuii strain S-52, the symbiotic partner of Astragalus sinicus

The O-specific polysaccharide (OPS) obtained by mild-acid degradation of the lipopolysaccharide isolated from Mesorhizobium huakuii strain S-52 was studied by sugar and ethylation analyses along with ¹H and ¹³C NMR spectroscopy. It was concluded that the OPS was composed of trisaccharide repeating units containing two residues of 6-deoxy-l-talose (6dTal) and one l-rhamnose (Rha), whose sequence in the OPS was determined by NOESY and HMBC experiments. The minor 3-O-acetylation (about 10%) of 6-deoxytalose glycosidically substituted at position-2 was judged by relative signal intensities of corresponding O-acetylated and non-acetylated 6dTal residues. Moreover, it was found that the non-reducing end of the OPS repeating unit was occupied by 3-O-methyl-d-fucose, which terminated the O-chain as a cap-residue. These data defined the structure of the OPS as:α-3-OMe-d-Fucp-(1→[2)-α-l-6dTalp-(1→3)-α-l-6dTalp-(1→2)-α-l-Rhap-(1→]_n     

l-Ribulose production from l-arabinose by an l-arabinose isomerase mutant from Geobacillus thermodenitrificans

Geobacillus thermodenitrificans, with a double-site mutation in L: -arabinose isomerase, produced 95 g L-: ribulose l(-1 ) from 500 g L: -arabinose l(-1) under optimum conditions of pH 8, 70 degrees C, and 10 units enzyme ml(-1) with a conversion yield of 19% over 2 h. The half-lives of the mutated enzyme at 70 and 75 degrees C were 35 and 4.5 h, respectively.     

Intracellular l-arginine concentration does not determine NO production in endothelial cells: Implications on the “l-arginine paradox”

We examined the relative contributory roles of extracellular vs. intracellular l-arginine (ARG) toward cellular activation of endothelial nitric oxide synthase (eNOS) in human endothelial cells. EA.hy926 human endothelial cells were incubated with different concentrations of ¹⁵N₄-ARG, ARG, or l-arginine ethyl ester (ARG-EE) for 2 h. To modulate ARG transport, siRNA for ARG transporter (CAT-1) vs. sham siRNA were transfected into cells. ARG transport activity was assessed by cellular fluxes of ARG, ¹⁵N₄-ARG, dimethylarginines, and l-citrulline by an LC–MS/MS assay. eNOS activity was determined by nitrite/nitrate accumulation, either via a fluorometric assay or by¹⁵N-nitrite or estimated ¹⁵N₃-citrulline concentrations when ¹⁵N₄-ARG was used to challenge the cells. We found that ARG-EE incubation increased cellular ARG concentration but no increase in nitrite/nitrate was observed, while ARG incubation increased both cellular ARG concentration and nitrite accumulation. Cellular nitrite/nitrate production did not correlate with cellular total ARG concentration. Reduced ¹⁵N₄-ARG cellular uptake in CAT-1 siRNA transfected cells vs. control was accompanied by reduced eNOS activity, as determined by ¹⁵N-nitrite, total nitrite and ¹⁵N₃-citrulline formation. Our data suggest that extracellular ARG, not intracellular ARG, is the major determinant of NO production in endothelial cells. It is likely that once transported inside the cell, ARG can no longer gain access to the membrane-bound eNOS. These observations indicate that the “l-arginine paradox” should not consider intracellular ARG concentration as a reference point.     

Cultivation ofCandida blankii in simulated bagasse hemicellulose hydrolysate

Summary All fourCandida blankii isolates evaluated for growth in simulated bagasse hemicellulose hydrolysate utilized the sugars and acetic acid completely. The utilization ofd-xylose,l-arabinose and acetic acid were delayed by the presence ofd-glucose, but after glucose depletion the other carbon sources were utilized simultaneously. The maximum specific growth rate of 0.36 h^–1 and cell yield of 0.47 g cells/g carbon source assimilate compared with published results obtained withC. utilis. C. blankii appeared superior toC. utilis for biomass production from hemicellulose hydrolysate in that it utilizedl-arabinose and was capable of growth at higher temperatures.     

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.     

Syntheses of dopa glycosides using glucosidases

Syntheses of l-dopa 1a glucoside 10a,b and dl-dopa 1b glycosides 10–18 with d-glucose 2, d-galactose 3, d-mannose 4, d-fructose 5, d-arabinose 6, lactose 7, d-sorbitol 8 and d-mannitol 9 were carried out using amyloglucosidase from Rhizopus mold, β-glucosidase isolated from sweet almond and immobilized β-glucosidase. Invariably, l-dopa and dl-dopa gave low to good yields of glycosides 10–18 at 12–49% range and only mono glycosylated products were detected through glycosylation/arylation at the third or fourth OH positions of l-dopa 1a and dl-dopa 1b. Amyloglucosidase showed selectivity with d-mannose 4 to give 4-O-C1β and d-sorbitol 8 to give 4-O-C6-O-arylated product. β-Glucosidase exhibited selectivity with d-mannose 4 to give 4-O-C1β and lactose 7 to give 4-O-C1β product. Immobilized β-glucosidase did not show any selectivity. Antioxidant and angiotensin converting enzyme inhibition (ACE) activities of the glycosides were evaluated glycosides, out of which l-3-hydroxy-4-O-(β-d-galactopyranosyl-(1′→4)β-d-glucopyranosyl) phenylalanine 16 at 0.9 ± 0.05 mM and dl-3-hydroxy-4-O-(β-d-glucopyranosyl) phenylalanine 11b,c at 0.98 ± 0.05 mM showed the best IC₅₀ values for antioxidant activity and dl-3-hydroxy-4-O-(6-d-sorbitol)phenylalanine 17 at 0.56 ± 0.03 mM, l-dopa-d-glucoside 10a,b at 1.1 ± 0.06 mM and dl-3-hydroxy-4-O-(d-glucopyranosyl)phenylalanine 11a-d at 1.2 ± 0.06 mM exhibited the best IC₅₀ values for ACE inhibition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Physiological comparison of d-cysteine desulfhydrase of Escherichia coli with 3-chloro-d-alanine dehydrochlorinase of Pseudomonas putida CR 1-1

d-Cysteine desulfhydrase of Escherichia coli W3110 trpED102/F trpED102 was physiologically characterized. It was found to be located in the cytosolic fraction, as 3-chloro-d-alanine dehydrochlorinase is. d-Cysteine desulfhydrase catalyzed not only the ,-elimination reaction of O-acetyl-d-serine to form pyruvate, acetic acid and ammonia, but also the -replacement reaction of O-acetyl-d-serine with sulfide to form d-cysteine. However, these reactions appeared not to proceed in vivo. No other activity of d-cysteine synthesis from O-acetyl-d-serine and sulfide was detected in a crude cell extract of E. coli which was immunotitrated with antibodies raised against the purified d-cysteine desulfhydrase. Although d-cysteine desulfhydrase catalyzes the degradation (,-elimination reaction) of 3-chloro-d-alanine, which is an effective antibacterial agent, E. coli W3110 trpED102/F trpED102 did not show resistance against 3-chloro-d-alanine. Therefore, d-cysteine desulfhydrase does not contribute to 3-chloro-d-alanine detoxification in vivo.