Two novel metal-independent long-chain alkyl alcohol dehydrogenases from Geobacillus thermodenitrificans NG80-2

Abstract

Two alkyl alcohol dehydrogenase (ADH) genes from the long-chain alkane-degrading strain Geobacillus thermodenitrificans NG80-2 were characterized in vitro. ADH1 and ADH2 were prepared heterologously in Escherichia coli as a homooctameric and a homodimeric protein, respectively. Both ADHs can oxidize a broad range of alkyl alcohols up to at least C 30 , as well as 1,3-propanediol and acetaldehyde. ADH1 also oxidizes glycerol, and ADH2 oxidizes isopropyl alcohol, isoamylol, acetone, octanal and decanal. The best substrate is ethanol for ADH1 and 1-octanol for ADH2. For both ADHs, the optimum assay condition is at 60 6C and pH 8.0, and both NAD and NADP can be used as the cofactor. Sequence analysis reveals that ADH1 and ADH2 belong to the Fe-containing/activated long-chain ADHs. However, the two enzymes contain neither Fe nor other metals, and Fe is not required for the activity, suggesting a new type of ADH. The ADHs characterized here are potentially useful in crude oil bioremediation and other bioconversion processes.

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

“…Finally, long-chain alcohol dehydrogenases (LDRs, such as the mannitol 2-dehydrogenase of Pseudomonas fluorescens), constitute a heterogeneous group that differ in the catalytic mechanism and additional cofactors needed (Zn, Fe, both, or no metal at all) [80,81]. Many employ a third catalytic mechanism different to those employed by SDR and MDR members, in which a Lys residue accepts a proton from the substrate, and then donates it to a solvent molecule.…”

Engineering of LadA for enhanced hexadecane oxidation using random- and site-directed mutagenesis

“…To determine the optimum pH, reactions were carried out at 60°C in the standard reaction mixture at different pH values ranging from pH 6.0 to pH 9.0, using KH 2 PO4-K 2 HPO4 buffer (for pH 6.0-8.0), Tris-HCl buffer (for pH 8.4), or glycine-NaOH buffer (for pH 8.6-9.0) buffer. To determine enzyme thermostability, reactions were incubated at 60°C for various periods prior to assaying enzyme activity under optimal conditions (Liu et al 2009). All the assays were based on both GC analysis of hexadecane disappearance and NADPH consumption in a spectrophotometer.…”

Crystallization and preliminary X-ray characterization of an NAD(P)-dependent butanol dehydrogenase A fromGeobacillus thermodenitrificansNG80-2

“…The expression and purification of gtADH2 were performed as described by Liu et al (2009) with some modifications. Briefly, the GTNG_2878 gene encoding gtADH2 (GenBank accession number: ABO68223.1) was cloned from G. thermodenitrificans NG80-2 and subcloned into a pET-28a(+) vector (Novagen) with a hexahistidine tag at the N-terminus.…”

“…NAD(P)-dependent ADHs can be divided into three major groups based on their molecular properties and metal content. Group I includes the Zn-containing 'long-chain' ADHs; group II includes 'short-chain' metal-free ADHs; and group III contains Fe-containing/ activated 'long-chain' ADHs (Liu et al, 2009). There has been increasing interest in studying ADHs (Radianingtyas & Wright, 2003), but their importance and the catalytic mechanism of iron in such enzymes are not well understood.…”

“…The first gene in the pathway, ladA, encodes a mono-oxygenase that serves as the key initiating enzyme, responsible for converting long-chain alkanes to the corresponding alkyl alcohols (Feng et al, 2007); its three-dimensional structure was reported by Li et al (2008). Two genes encode alcohol dehydrogenases (adh1 and adh2) for the conversion of alkyl alcohols to the corresponding alkyl aldehydes (Liu et al, 2009). The final gene in the alkane degradation pathway, aldh, encodes an aldehyde dehydrogenase for the conversion of alkyl aldehydes to fatty acids.…”