Artificial Thermostable D-Amino Acid Dehydrogenase: Creation and Application.

Hironaga Akita,Haruhiko Sakuraba,Junji Hayashi,Toshihisa Ohshima

doi:10.3389/fmicb.2018.01760

Abstract

Many kinds of NAD(P)+-dependent L-amino acid dehydrogenases have been so far found and effectively used for synthesis of L-amino acids and their analogs, and for their sensing. By contrast, similar biotechnological use of D-amino acid dehydrogenase (D-AADH) has not been achieved because useful D-AADH has not been found from natural resources. Recently, using protein engineering methods, an NADP+-dependent D-AADH was created from meso-diaminopimelate dehydrogenase (meso-DAPDH). The artificially created D-AADH catalyzed the reversible NADP+-dependent oxidative deamination of D-amino acids to 2-oxo acids. The enzyme, especially thermostable one from thermophiles, was efficiently applicable to synthesis of D-branched-chain amino acids (D-BCAAs), with high yields and optical purity, and was useful for the practical synthesis of 13C- and/or 15N-labeled D-BCAAs. The enzyme also made it possible to assay D-isoleucine selectively in a mixture of isoleucine isomers. Analyses of the three-dimensional structures of meso-DAPDH and D-AADH, and designed mutations based on the information obtained made it possible to markedly enhance enzyme activity and to create D-AADH homologs with desired reactivity profiles. The methods described here may be an effective approach to artificial creation of biotechnologically useful enzymes.

Highlights

With the exception of glycine, proteinogenic α-amino acids all contain an asymmetrical carbon, resulting in the occurrence of D- and L-enantiomers
From multiple sequence alignment of mesoDAPDH genes from U. thermosphaericus and several other bacterial strains, we found that the five amino acid residues (Gln150, Asp154, Thr169, Arg195, and His244) mutated in the C. glutamicum enzyme to create a D-amino acid dehydrogenase (D-AADH) were completely conserved in the sequence of the U. thermosphaericus enzyme
For one step production of D-branched-chain amino acids (D-BCAAs) from the corresponding 2-oxo acids, we developed a two enzyme-coupled system composed of D-AADH and glucose dehydrogenase (GDH) from the thermoacidophilic crenarchaeon Sulfolobus tokodaii (Figure 2A; Ohshima et al, 2003)

Summary

INTRODUCTION

With the exception of glycine, proteinogenic α-amino acids all contain an asymmetrical carbon, resulting in the occurrence of D- and L-enantiomers. They showed that the engineered enzyme was able to produce several D-amino acids through reductive amination of the corresponding 2oxo acids This D-AADH prepared from mesophilic C. glutamicum meso-DAPDH was not sufficiently stable for use as the catalyst in a bioreactor. Vedha-Peters et al (2006) prepared an NADP+-dependent D-AADH by introducing five substitutions at the active site of the C. glutamicum meso-DAPDH This mutant enzyme is capable of one-step production of several L-amino acids via reductive amination of the corresponding 2-oxo acids with ammonia and NADPH. Because this enzyme was prepared from a mesophilic bacterial meso-DAPDH, it is not sufficiently stable for use under the conditions necessary for industrial application. 2-Oxooctanoate Phenylpyruvate Pyruvate 2-Oxobutanoate 2-Oxohexanoate 2-Oxopentanoate 2-Oxo-3-methylbutanoate 2-Oxo-3-methylpentanoate 2-Oxo-4-methylpentanoate 2-Oxo-4-methylthio butanoate

Reductive amination

Findings

INSIGHT INTO THE COENZYME AND SUBSTRATE RECOGNITION MECHANISMS