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

Abstract

BackgroundThe 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.ResultsThe 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).ConclusionThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0899-9) contains supplementary material, which is available to authorized users.

Two enzymes of the acetone degradation pathway of Desulfococcus biacutus: coenzyme B12‐dependent 2‐hydroxyisobutyryl‐CoA mutase and 3‐hydroxybutyryl‐CoA dehydrogenase

“…The previously identified zinc-dependent dehydrogenase (strongly induced during growth with acetone, and encoded in a different gene cluster), which catalyzes NAD 1 /NADH-dependent oxidations/reductions of a wide range of short-chain alcohols/aldehydes, e.g., reduction of 3-hydroxybutanal, may help to sequester side products formed during acetone activation, for example during CoA cofactor limitation or due to unspecific reactions forming reactive aldehydes (Fig. 1A) (Guti errez Acosta et al, 2014;Frey et al, 2016).…”

“…Analysis of protein expression and protein purification was done by polyacrylamide gel electrophoresis (1D‐PAGE) or native PAGE (Frey et al ., ); proteins were stained with colloidal Coomassie Brilliant Blue (Neuhoff et al ., ). Proteins were identified by peptide fingerprinting mass spectrometry at the Proteomics facility of University of Konstanz (Gutiérrez Acosta et al ., ).…”

“…The polymerase used was Phusion High-Fidelity DNA Polymerase (New England Biolabs), and the PCR conditions were 35 cycles of 45 s denaturation at 988C, 45 s annealing at 608C (658C for 04573 and 04574) and elongation at 728C depending on product length (1 min per 1000 bp). The PCR products were inserted either into the expression vector pET100 (N-terminal His 6 -tag; for Debia-HcmB) or pET101 (C-terminal His 6 -tag; for Debia-ACPR and Debia-HcmA) of the Champion pET Directional TOPO Expression Kit (Invitrogen) Acetone activation in sulfate-reducing bacteria 289 (Frey et al, 2016). Plasmid DNA was purified from E. coli using the Zymo MiniPrep Kit (Zymo Research), and used for sequencing (GATC Biotech, Germany).…”

“…Plasmid DNA was purified from E. coli using the Zymo MiniPrep Kit (Zymo Research), and used for sequencing (GATC Biotech, Germany). Transformation of chemically competent E. coli-Rosetta 2 (DE3) cells (Merck KGaA, Germany) was performed, as described previously (Frey et al, 2016). Primer design and sequence data analysis was accomplished using the DNASTAR Lasergene software package.…”

“…However, genome sequencing and differential proteomics comparing acetone-and butyrate-grown cells (Guti errez Acosta et al, 2014) identified several proteins that are specifically induced during growth with acetone. One of these (IMG locus tag DebiaDRAFT_04514) has recently been characterized as a zinc-dependent alcohol dehydrogenase capable of converting a wide range of aldehydes and alcohols (Frey et al, 2016). Further, in a different gene cluster, genes of four strongly acetone-inducible proteins annotated as a predicted TDPdependent enzyme (DebiaDRAFT_04566), two subunits of a B 12 -dependent methylmalonyl-CoA mutase-like enzyme (DebiaDRAFT_04573 and 04574), and a predicted shortchain alcohol dehydrogenase (DebiaDRAFT_04573), were also identified by proteomics (Guti errez Acosta et al, 2014).…”