Cloning, functional expression and characterization of a bifunctional 3-hydroxybutanal dehydrogenase /reductase involved in acetone metabolism by Desulfococcus biacutus

Abstract

Background<br /><br />The strictly anaerobic, sulfate-reducing bacterium Desulfococcus biacutus can utilize acetone as sole carbon and energy source for growth. Whereas in aerobic and nitrate-reducing bacteria acetone is activated by carboxylation with CO₂ to acetoacetate, D. biacutus involves CO as a cosubstrate for acetone activation through a different, so far unknown pathway. Proteomic studies indicated that, among others, a predicted medium-chain dehydrogenase/reductase (MDR) superfamily, zinc-dependent alcohol dehydrogenase (locus tag DebiaDRAFT_04514) is specifically and highly produced during growth with acetone.<br /><br />Results<br /><br />The MDR gene DebiaDRAFT_04514 was cloned and overexpressed in E. coli. The purified recombinant protein required zinc as cofactor, and accepted NADH/NAD⁺ but not NADPH/NADP⁺ as electron donor/acceptor. The pH optimum was at pH 8, and the temperature optimum at 45 °C. Highest specific activities were observed for reduction of C₃ - C₅-aldehydes with NADH, such as propanal to propanol (380 ± 15 mU mg⁻¹ protein), butanal to butanol (300 ± 24 mU mg⁻¹), and 3-hydroxybutanal to 1,3-butanediol (248 ± 60 mU mg⁻¹), however, the enzyme also oxidized 3-hydroxybutanal with NAD⁺ to acetoacetaldehyde (83 ± 18 mU mg⁻¹).<br /><br />Conclusion<br /><br />The enzyme might play a key role in acetone degradation by D. biacutus, for example as a bifunctional 3-hydroxybutanal dehydrogenase/reductase. Its recombinant production may represent an important step in the elucidation of the complete degradation pathway.