NADP+-dependent l-arginine dehydrogenase from Pseudomonas velonii: Purification, characterization and application to an l-arginine assay
Introduction
More than twenty different amino acid dehydrogenases (EC 1.4.1.-), catalyzing the reversible deamination of amino acids to their corresponding oxo analogs in the presence of NAD(P)+, have been found so far. Among them, glutamate dehydrogenase, leucine dehydrogenase, alanine dehydrogenase, phenylalanine dehydrogenase and aspartate dehydrogenase have all been well characterized and are widely used for industrial asymmetric production of l-amino acids and their analogs [[1], [2], [3], [4], [5]], which then serve as important nutrients and pharmaceutical compounds. In addition, amino acid dehydrogenases have been used for sensing amino acids in foods and blood, and for enzyme assays [1,6,7]. For those reasons, novel amino acid dehydrogenases have the potential to be highly useful like the case of artificially prepared d-amino acid dehydrogenases [[8], [9], [10], [11], [12]]. Among these enzymes, NAD(P)+-dependent l-arginine dehydrogenase (EC 1.4.1.25, L-ArgDH) is one of the most recently found and is known to physiologically function in concert with FAD-dependent d-arginine dehydrogenase for the conversion (isomerization) of d-arginine to l-arginine via its oxo-analog, 5-guanidino-2-oxopentanoate, in Psuedomonas aeruginosa POA1 [13]. The gene DauB in P. aeruginosa has been expressed in Escherichia coli, and the product reportedly catalyzes the reversible deamination of l-arginine to 5-guanidino-2-oxopentanoate in the presence of NAD(P)+. However, the molecular and catalytic properties of L-ArgDH have not yet been reported due to its instability.
We previously identified a putative L-ArgDH gene, PverR02_12350, within the genome of the nonpathogenic bacterium P. veronii (KEGG Organisms: Complete Genomes: www.genome.jp/kegg/catalog/org list. Html). This gene possesses amino acid sequence homology (67.8%) with a gene encoding L-ArgDH in the pathogenic bacterium P. aeruginosa POA1. Here, we succeeded in expressing the P. veronii JCM 11942 gene in E. coli, using artificial primers synthesized from the N- and C-terminal sequences inferred from the putative L-ArgDH gene. The expressed product was purified, characterized, and applied to a simple colorimetric assay for l-arginine.
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Materials
l-Arginine, NADP+and NAD+ were purchased from Wako Pure Chemical Corporation (Tokyo, Japan). Methoxy-phenasine methosulfate (m-PMS), water soluble tetrazolium 1 (WST-1) and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT) were from Chemical Dojin Co., Ltd. (Kumamoto, Japan). Yeast extract and Luria-Bertani (LB) medium were from Becton, Dickinson and Company (Sparks, MD, USA), and hipolypepton S was from Nihon Pharmaceutical Co., Ltd. (Tokyo). P. veronii JCM 11942 was
Expression, stabilization, and purification of NADP+-dependent L-ArgDH
A putative gene encoding L-ArgDH in the nonpathogenic bacterium P. veronii JCM 11942 was successively expressed in E. coli cells, after which the cell extract exhibited strong NADP+-dependent L-ArgDH activity. However, the enzyme in the crude extract containing 20 mM phosphate buffer, pH 7.9 or 7.2, supplemented with 0.01% 2-MET was somewhat unstable, and nearly all activity was lost within 2 days (Fig. 1). We therefore evaluated the stabilizing effect of adding glycerol to the enzyme solution
Data availability
The data underlying this article will be shared on reasonable request to the corresponding author.
Author contribution
All authors contributed to the study conception and design, and performed material preparation, data collection and analysis. T. Ohshima mainly wrote the manuscript in discussion during the preparation of the manuscript.
Funding
This work was supported in part by research funding from the Japan Society for the Promotion of Science KAKENHI grant No. 20K05816 to T. O.
Declaration of competing interest
No potential conflict of interest was reported by the authors.
Acknowledgments
We thank William Goldman, Ph.D. (MST Editing Company, Baltimore, USA) for English language editing.
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