Expression of the NAD-dependent FDH1 β-subunit from Methylobacterium extorquens AM1 in Escherichia coli and its characterization

• Published in: Biotechnology and bioprocess engineering Vol. 19; no. 4; pp. 613 - 620
• Main Authors: Choe, Hyunjun, Lee, Sumi, Hwang, Hyojin, Joo, Jeong Chan, Cho, Dae Haeng, Kim, Yong Hwan
• Format: Journal Article
• Language: English
• Published: Heidelberg Springer-Verlag 01-07-2014; The Korean Society for Biotechnology and Bioengineering; 한국생물공학회
• Subjects: Biotechnology; catalytic activity; Chemistry; Chemistry and Materials Science; electron transfer; enzyme activity; Escherichia coli; Industrial and Production Engineering; industrial applications; Methylobacterium extorquens; NAD (coenzyme); nicotinamide; oxidation; oxygen; paraquat; Research Paper; 생물공학; formate dehydrogenase; AM1; electron mediator; iron-sulfur cluster; NADH oxidase
• ISSN: 1226-8372; 1976-3816
• DOI: 10.1007/s12257-014-0126-1

The efficient regeneration of nicotinamide cofactors is an important process for industrial applications because of their high cost and stoichiometric requirements. In this study, the FDH1 β-subunit of NAD-dependent formate dehydrogenase from Methylobacterium extorquens AM1 was heterologously expressed in Escherichia coli. It showed water-forming NADH oxidase (NOX-2) activity in the absence of its α-subunit. The β-subunit oxidized NADH and generated NAD⁺. The enzyme showed a low NADH oxidation activity (0.28 U/mg enzyme). To accelerate electron transfer from the enzyme to oxygen, four electron mediators were tested; flavin mononucleotide, flavin adenine dinucleotide, benzyl viologen (BV), and methyl viologen. All tested electron mediators increased enzyme activity; addition of 250 μM BV resulted in the largest increase in enzyme activity (9.98 U/mg enzyme; a 35.6-fold increase compared with that in the absence of an electron mediator). Without the aid of an electron mediator, the enzyme had a substrate-binding affinity for NADH (K ₘ) of 5.87 μM, a turnover rate (k cₐₜ) of 0.24/sec, and a catalytic efficiency (k cₐₜ/K ₘ) of 41.31/mM/sec. The addition of 50 μM BV resulted in a 22.75-fold higher turnover rate (k cₐₜ, 5.46/sec) and a 2.64-fold higher catalytic efficiency (k cₐₜ/K ₘ, 107.75/mM/sec).