Development of β-Amino Acid Dehydrogenase for the Synthesis of β-Amino Acids via Reductive Amination of β-Keto Acids

ACS Catalysis ◽  
2015 ◽  
Vol 5 (4) ◽  
pp. 2220-2224 ◽  
Author(s):  
Dalong Zhang ◽  
Xi Chen ◽  
Rui Zhang ◽  
Peiyuan Yao ◽  
Qiaqing Wu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Reductive Amination ◽  
Acid Dehydrogenase ◽  
Amino Acid Dehydrogenase ◽  
Keto Acids

scholarly journals Creation of a Broad-Range and Highly Stereoselectived-Amino Acid Dehydrogenase for the One-Step Synthesis ofd-Amino Acids

2006 ◽  
Vol 128 (33) ◽  
pp. 10923-10929 ◽  
Author(s):  
Kavitha Vedha-Peters ◽  
Manjula Gunawardana ◽  
J. David Rozzell ◽  
Scott J. Novick
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Acid Dehydrogenase ◽  
Amino Acid Dehydrogenase ◽  
One Step ◽  
The One

scholarly journals Regulation of the dauBAR operon and characterization of d-amino acid dehydrogenase DauA in arginine and lysine catabolism of Pseudomonas aeruginosa PAO1

Microbiology ◽  
2010 ◽  
Vol 156 (1) ◽  
pp. 60-71 ◽  
Author(s):  
Congran Li ◽  
Xiangyu Yao ◽  
Chung-Dar Lu
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Mutation Analysis ◽  
Regulatory Region ◽  
Mobility Shift ◽  
Signal Molecule ◽  
Acid Dehydrogenase ◽  
Amino Acid Dehydrogenase ◽  
Activity Measurements

A unique d-to-l racemization of arginine by coupled arginine dehydrogenases DauA and DauB encoded by the dauBAR operon has been recently reported as a prerequisite for d-arginine utilization as the sole source of carbon and nitrogen through l-arginine catabolic pathways in P. aeruginosa. In this study, enzymic properties of the catabolic FAD-dependent d-amino acid dehydrogenase DauA and the physiological functions of the dauBAR operon were further characterized with other d-amino acids. These results establish DauA as a d-amino acid dehydrogenase of broad substrate specificity, with d-Arg and d-Lys as the two most effective substrates, based on the kinetic parameters. In addition, expression of dauBAR is specifically induced by exogenous d-Arg and d-Lys, and mutations in the dauBAR operon affect utilization of these two amino acids alone. The function of DauR as a repressor in the control of the dauBAR operon was demonstrated by dauB promoter activity measurements in vivo and mobility shift assays with purified His-tagged protein in vitro. The potential effect of 2-ketoarginine (2-KA) derived from d-Arg deamination by DauA as a signal molecule in dauBAR induction was first revealed by mutation analysis and further supported by its in vitro effect on alleviation of DauR–DNA interactions. Through sequence analysis, putative DauR operators were identified and confirmed by mutation analysis. Induction of the dauBAR operon to the maximal level was found to require the l-arginine-responsive regulator ArgR, as supported by the loss of inductive effect by l-Arg on dauBAR expression in the argR mutant and binding of purified ArgR to the dauB regulatory region in vitro. In summary, this study establishes that optimal induction of the dauBAR operon requires relief of DauR repression by 2-KA and activation of ArgR by l-Arg as a result of d-Arg racemization by the encoded DauA and DauB.

scholarly journals Electrochemical Molecular Conversion of α-Keto Acid to Amino Acid at a Low Overpotential Using a Nanoporous Gold Catalyst

2021 ◽  
Vol 22 (17) ◽  
pp. 9442
Author(s):  
Yasuhiro Mie ◽  
Shizuka Katagai ◽  
Chitose Mikami
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gold Electrode ◽  
Reductive Amination ◽  
Gold Catalyst ◽  
Nanoporous Gold ◽  
Keto Acid ◽  
Applied Potential ◽  
Keto Acids ◽  
Optimized Conditions

A nanoporous gold (NPG) electrode prepared through a facile anodization technique was employed in the electrochemical reductive amination of biomass-derivable α-keto acids in the presence of a nitrogen source to produce the corresponding amino acids. NPG showed a clear reductive current in the presence of α-keto acid and NH2OH, and the electrolysis experiments confirmed the production of L-amino acid. A reductive voltammetric signal at the NPG electrode appeared at a more positive potential by 0.18–0.79 V, compared with those at the planar-gold electrode without anodization and other previously reported electrode systems, indicating the high activity of the prepared nanostructure for the electrochemical reaction. Maximum Faradaic efficiencies (FEs) of 74–93% in the reductive molecular conversion to amino acids of Ala, Asp, Glu, Gly, and Leu were obtained under the optimized conditions. The FE values were strongly dependent on the applied potential in the electrolysis, suggesting that the hydrogen evolution reaction at the electrode surface was more significant as the applied potential became more negative. The effect of potential at the NPG was lower than that at the planar-gold electrode. These results indicate that nanostructurization decreases the overpotential for the electrochemical reductive amination, resulting in high FE.

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

2017 ◽  
Vol 83 (11) ◽  
Author(s):  
Junji Hayashi ◽  
Tomonari Seto ◽  
Hironaga Akita ◽  
Masahiro Watanabe ◽  
Tamotsu Hoshino ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Specific Activity ◽  
High Specific Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
High Catalytic Activity ◽  
Acid Dehydrogenase ◽  
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.

scholarly journals Interference by branched-chain amino acids in the assay of alanine with alanine dehydrogenase

1978 ◽  
Vol 174 (3) ◽  
pp. 1079-1082 ◽  
Author(s):  
P Lund ◽  
G Baverel
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Dehydrogenase Activity ◽  
Branched Chain Amino Acids ◽  
Acid Dehydrogenase ◽  
Alanine Dehydrogenase ◽  
Amino Acid Dehydrogenase ◽  
Branched Chain Amino Acid ◽  
Branched Chain

Commercial preparations of alanine dehydrogenase from Bacillus subtilis are contaminated to varying extents with activity towards branched-chain amino acids. The Km values for these amino acids are of the same order as for L-alanine (about 10(-3)M). The branched-chain amino acid dehydrogenase activity is lost on dialysis for 2–4h against water or 2mM-EDTA.

Enzymatic synthesis of cyclic amino acids by N-methyl-l-amino acid dehydrogenase from Pseudomonas putida

2006 ◽  
Vol 17 (12) ◽  
pp. 1775-1779 ◽  
Author(s):  
Mari Yasuda ◽  
Makoto Ueda ◽  
Hisashi Muramatsu ◽  
Hisaaki Mihara ◽  
Nobuyoshi Esaki
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Pseudomonas Putida ◽  
Enzymatic Synthesis ◽  
Acid Dehydrogenase ◽  
Amino Acid Dehydrogenase ◽  
Cyclic Amino Acids

scholarly journals Dye-linked d-Proline Dehydrogenase from Hyperthermophilic ArchaeonPyrobaculum islandicumIs a Novel FAD-dependent Amino Acid Dehydrogenase

2002 ◽  
Vol 277 (15) ◽  
pp. 12861-12867 ◽  
Author(s):  
Takenori Satomura ◽  
Ryushi Kawakami ◽  
Haruhiko Sakuraba ◽  
Toshihisa Ohshima
Keyword(s):  
Amino Acid ◽  
Proline Dehydrogenase ◽  
Acid Dehydrogenase ◽  
Amino Acid Dehydrogenase
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