Molecular characterization of two Clostridium acetobutylicum ATCC 824 butanol dehydrogenase isozyme genes

Abstract

A 4-kb segment of DNA containing two previously cloned butanol dehydrogenase (BDH) isozyme genes (D.Petersen, R. Welch, F. Rudolph, and G. Bennett, J. Bacteriol. 173:1831-1834 was sequenced. Two complete open reading frames (ORFs) were identified (bdhA and bdhB), along with a third truncated ORF (ORF1). The translation products of bdka4 and bdhB corresponded to the N-terminal sequences of the purified BDH I and BDH H proteins, respectively. The two isozymes had a high amino acid identity (73%) and showed homology to a newly described class of alcohol dehydrogenases. Northern blots revealed that bdhA and bdhB did not form an operon. Primer extension experiments located single transcriptional start sites 37 and 58 bp upstream of the start codons of bdhA and bdhB, respectively. The -10 and -35 promoter regions for these genes were almost identical. bMUA and bdhB were found to be induced or derepressed immediately prior to significant butanol production in controlled pH 5.0 batch fermentations.The gram-positive, obligately anaerobic bacterium Clostridium acetobutylicum is one of the few organisms known to produce 1-butanol as a major fermentation product. Interest in the use of butanol as a fuel extender and chemical feedstock has prompted several investigations into the molecular means by which C acetobutylicum produces butanol.The butanol dehydrogenase (BDH) is involved in the final step of the butanol formation pathway in C. acetobutylicum. In this step, butyraldehyde is converted to butanol, with the cofactor NAD(P)H being oxidized in the process. Evidence suggests that there are two distinct types of BDHs in C. acetobutylicum, one which is NADH dependent and one that is NADPH dependent (11,25). The relative physiological importance of these two dehydrogenases is unclear at present. Previously, Youngleson et al. (42,43) cloned and sequenced the adhl gene from C. acetobutylicum P262. This gene codes for a 43-kDa NADPH-dependent alcohol dehydrogenase (ADH). No activity was observed with NADH as a cofactor. The enzyme utilized ethanol and butanol nearly equally well and was thus classified as an ADH.More recently, two BDH isozymes (BDH I, BDH II) were purified and cloned from C. acetobutylicum ATCC 824 (25,35,36). These isozymes were primarily NADH dependent, although some NADPH-dependent activity was observed. Both enzymes were dimers with a subunit molecular size of -42 kDa. The BDH II enzyme was found to have 46-fold greater activity with butyraldehyde than with acetaldehyde, whereas the BDH I enzyme had only 2-fold greater activity.In this study, the nucleotide sequence and putative amino acid sequence of the bdhA and bdhB genes are presented.These two genes code for proteins showing homology to a newly described class of ADHs. Although the bdhA and bdhB genes were found to exist in tandem on the chromosome, RNA studies revealed that they do not form an operon. In addition, both genes were shown to be induced or * Corresponding author. derepressed near the onset of butanol formation in batch culture. MATERIALS ...

Coenzyme A-transferase-independent butyrate re-assimilation in Clostridium acetobutylicum—evidence from a mathematical model

“…The concerted action of a putative aldehyde:ferredoxin oxidoreductase (AOR, CAC2018) and NADH-dependent butanol dehydrogenases, BdhA and BdhB (Petersen et al 1991;Welch et al 1989), could even bypass the indermediate butyryl-CoA and result in an immediate increase of butanol as a direct stress response to the rapidly changing pH level. Whereas bdhA and bdhB are arranged in monocistronic operons in the chromosome, in each case controlled by a single promoter (Walter et al 1992), computational predictions using the published annotated genome (Nölling et al 2001) suggest that AOR forms an operon in the clostridial chromosome that includes several enzymes for fatty acids biosynthesis, two enoyl-CoA hydratases, and a 3-hydroxyacyl-CoA dehydrogenase (Caspi et al 2012;Dale et al 2010;Karp et al 2010). Importantly, the butanol dehydrogenases possess much higher activity with butyraldehyde than with acetaldehyde, dropping sharply from their maximum at pH=5.5 to less than 50% with changes of ±0.7 pH unit (Petersen et al 1991).…”

“…The gene product of adhE2 is induced during acidogenesis (Grimmler et al 2011) and is believed to facilitate only the formation of ethanol. In contrast, AdhE1 catalyses the production of both ethanol and butanol Walter et al 1992) and is induced during solventogenesis (Fontaine et al 2002;Grimmler et al 2011). On the basis of these experimental findings, the model considers that acidogenic and solventogenic cells differ in their proteomic composition, including both AdhE1 and AdhE2, see Eqs.…”

Effects of elemental sulfur on the metabolism of the deep-sea hyperthermophilic archaeon Thermococcus strain ES-1: characterization of a sulfur-regulated, non-heme iron alcohol dehydrogenase

“…This suggests that T. litoralis ADH is also an iron-containing enzyme but that it loses iron during purification. A diverse multitude of microbial NAD(P)-dependent ADHs are known to exist (recently reviewed in reference 54), but only a limited number fall into the so-called group III family, the members of which are characterized by their primary structure and subunit size and by a requirement for a divalent metal ion for activity (21,24,26,30,33,73). Of these, only the enzymes from Zymomonas mobilis (ADHII [49,63]) and E. coli (adhE [26,33]) have been shown to be iron dependent, and in both cases the metal is lost during purification (32,49).…”

Differentiation-dependent expression of Adhfe1 in adipogenesis

“…Although this new type of ADH lacked homology with any other previously known ADHs [29], they were demonstrated to utilize ethanol, as well as number of other alcohols, as substrate(s) [6]. In the intervening years, a number of additional microbial enzymes of this iron-activated enzyme type have been described [11,32,50,54,73,83]. Despite the nomenclature of "iron-activated" ADHs, these enzymes are variously activated by a range of divalent cations.…”