Characterization of NADP-dependent L-arginine dehydrogenase as a novel amino acid dehydrogenase and its application to an L-arginine assay

L-Arginine dehydrogenase (L-ArgDH, EC 1.4.1.25) is an amino acid dehydrogenase which catalyzes the reversible oxidative deamination of L-arginine to the oxo analog in the presence of NADP. Although the enzyme activity is detected in the cell extract of Pseudomonas aruginosa , the purification and characterization of the enzyme have not been achieved to date. We here found the gene homolog of L-ArgDH in genome data of Pseudomonas veronii and succeeded in expression of P. veronii JCM11942 gene in E. coli. The gene product exhibited strong NADP-dependent L-ArgDH activity. The crude enzyme was unstable under neutral pH conditions, but was markedly stabilized by the addition of 10% glycerol. The enzyme was purified to homogeneity through a single Ni-chelate affinity ch romatography step and consisted of a homodimeric protein with a molecular mass of about 65 kDa. The enzyme selectively catalyzed l-arginine oxidation in the presence of NADP with maximal activity at pH 9.5. The apparent K m values for l-arginine and NADP were 2.5 and 0.21 mM, respectively. The nucleotide sequence coding the enzyme gene ( was determined and the amino acid sequence was deduced from the nucleotide sequence. As an application of the enzyme, simple colorimetric microassay for L-arginine using the enzyme was achieved.

The Capability of Utilizing Abiotic Enantiomers of Amino Acids by Halomonas sp. LMO_D1 Derived From the Mariana Trench

D-amino acids (D-AAs) have been produced both in organisms and in environments via biotic or abiotic processes. However, the existence of these organic materials and associated microbial degradation activity has not been previously investigated in subduction zones where tectonic activities result in the release of hydrothermal organic matter. Here, we isolated the bacterium Halomonas sp. LMO_D1 from a sample obtained from the Mariana trench, and we determined that this isolate utilized 13 different D-AAs (D-Ala, D-Glu, D-Asp, D-Ser, D-Leu, D-Val, D-Tyr, D-Gln, D-Asn, D-Pro, D-Arg, D-Phe, and D-Ile) in the laboratory and could grow on D-AAs under high hydrostatic pressure (HHP). Moreover, the metabolism of L-AAs was more severely impaired under HHP conditions compared with that of their enantiomers. The essential function gene (Chr_2344) required for D-AA catabolism in strain LMO_D1 was identified and confirmed according to the fosmid library method used on the D-AAs plate. The encoded enzyme of this gene (DAADH_2344) was identified as D-amino acid dehydrogenase (DAADH), and this gene product supports the catabolism of a broad range of D-AAs. The ubiquitous distribution of DAADHs within the Mariana Trench sediments suggests that microorganisms that utilize D-AAs are common within these sediments. Our findings provide novel insights into the microbial potential for utilizing abiotic enantiomers of amino acids within the subduction zone of the Mariana trench under HHP, and our results provide an instructive significance for understanding these abiotic enantiomers and allow for insights regarding how organisms within extraterrestrial HHP environments can potentially cope with toxic D-AAs.

Characterization of NADP-dependent L-arginine dehydrogenase as a novel amino acid dehydrogenase and its application to an L-arginine assay

Purpose: The primary aim of this study was the purification and characterization of an NADP-dependent L arginine dehydrogenase (L-ArgDH, EC 1.4.1.25) as a novel amino acid dehydrogenase from Pseudomonas veronii. We then applied the enzyme to an L-arginine assay. Methods: An L-ArgDH gene from P. veronii JCM11942 was amplified by PCR using primers based on the N and C-terminal sequences inferred from a putative L-ArgDH gene (PverR02_12350) found in the P. veronii genome. The L-ArgDH activity of the product expressed in Escherichia coli was confirmed, after which the enzyme was purified, characterized, and applied to an L-Arg microassay. Results: The P. veroniiJCM11942 gene was expressed in E. coli, and the gene product exhibited strong NADP dependent L-ArgDH activity. The crude enzyme was unstable but was stabilized by the presence of 10% glycerol under neutral pH conditions. The enzyme was purified to homogeneity through a single Ni-chelate affinity chromatography step and consisted of a homodimeric protein with a molecular mass of about 65 kDa. The enzyme selectively catalyzed L-arginine oxidation in the presence of NADP, with maximal activity at pH 9.5. The apparent Km values for L-arginine and NADP were 2.5 and 0.21 mM, respectively. A simple colorimetric microassay for L-arginine was achieved using the enzyme. Conclusions: The L-ArgDH gene from P. veronii JCM 11942 was successively expressed in E. coli. The product exhibited NADP-dependent L-ArgDH dehydrogenase activity, and the enzyme was purified and characterized as a novel amino acid dehydrogenase. Furthermore, a simple colorimetric assay for L-arginine using L-ArgDH was achieved. Conflict of interest: The authors declare that they have no competing interests.

L-Alanine Exporter, AlaE, of Escherichia coli Functions as a Safety Valve to Enhance Survival under Feast Conditions

The intracellular level of amino acids is determined by the balance between their anabolic and catabolic pathways. L-alanine is anabolized by three L-alanine synthesizing enzymes and catabolized by two racemases and D-amino acid dehydrogenase (DadA). In addition, its level is regulated by L-alanine movement across the inner membrane. We identified the novel gene alaE, encoding an L-alanine exporter. To elucidate the physiological function of L-Alanine exporter, AlaE, we determined the susceptibility of alaE-, dadA-, and alaE/dadA-deficient mutants, derived from the wild-type strain MG1655, to L-alanyl-L-alanine (Ala-Ala), which shows toxicity to the L-alanine-nonmetabolizing variant lacking alaE. The dadA-deficient mutant has a similar minimum inhibitory concentration (MIC) (>1.25 mg/mL) to that observed in MG1655. However, alaE- and alaE/dadA-deficient mutants had MICs of 0.04 and 0.0025 mg/mL, respectively. The results suggested that the efficacy of AlaE to relieve stress caused by toxic intracellular accumulation of L-alanine was higher than that of DadA. Consistent with this, the intracellular level of alanine in the alaE-mutant was much higher than that in MG1655 and the dadA-mutant. We, therefore, conclude that AlaE functions as a ‘safety-valve’ to prevent the toxic level accumulation of intracellular L-alanine under a peptide-rich environment, such as within the animal intestine.

Structure-Based Engineering of an Artificially Generated NADP+-Dependent d-Amino Acid Dehydrogenase

ABSTRACT A stable NADP+-dependent d-amino acid dehydrogenase (DAADH) was recently created from Ureibacillus thermosphaericus meso-diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 Å, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized d-phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 μmol/min/mg at 50°C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward d-leucine, d-norleucine, d-methionine, d-isoleucine, and d-tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 μmol/min/mg at 50°C). The structures of the D94A-Y224F double mutant in complex with NADP+ and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxo-analogue) were then determined at resolutions of 1.59 Å and 1.74 Å, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic d-amino acids with bulky side chains. IMPORTANCE In recent years, the potential uses for d-amino acids as source materials for the industrial production of medicines, seasonings, and agrochemicals have been growing. To date, several methods have been used for the production of d-amino acids, but all include tedious steps. The use of NAD(P)+-dependent d-amino acid dehydrogenase (DAADH) makes single-step production of d-amino acids from oxo-acid analogs and ammonia possible. We recently succeeded in creating a stable DAADH and demonstrated that it is applicable for one-step synthesis of d-amino acids, such as d-leucine and d-isoleucine. As the next step, the creation of an enzyme exhibiting different substrate specificity and higher catalytic efficiency is a key to the further development of d-amino acid production. In this study, we succeeded in creating a novel mutant exhibiting extremely high catalytic activity for phenylpyruvate amination. Structural insight into the mutant will be useful for further improvement of DAADHs.