Abstract
A non-bifurcating NADH-dependent, dimeric [FeFe]-hydrogenase (HydAB) from Syntrophus aciditrophicus was heterologously produced in Escherichia coli, purified and characterized. Purified recombinant HydAB catalyzed NAD+ reduction coupled to hydrogen oxidation and produced hydrogen from NADH without the involvement of ferredoxin. Hydrogen partial pressures (2.2–40.2 Pa) produced by the purified recombinant HydAB at NADH to NAD+ ratios of 1–5 were similar to the hydrogen partial pressures generated by pure and cocultures of S. aciditrophicus (5.9–36.6 Pa). Thus, the hydrogen partial pressures observed in metabolizing cultures and cocultures of S. aciditrophicus can be generated by HydAB if S. aciditrophicus maintains NADH to NAD+ ratios greater than one. The flavin-containing beta subunits from S. aciditrophicus HydAB and the non-bifurcating NADH-dependent S. wolfei Hyd1ABC share a number of conserved residues with the flavin-containing beta subunits from non-bifurcating NADH-dependent enzymes such as NADH:quinone oxidoreductases and formate dehydrogenases. A number of differences were observed between sequences of these non-bifurcating NADH-dependent enzymes and [FeFe]-hydrogenases and formate dehydrogenases known to catalyze electron bifurcation including differences in the number of [Fe-S] centers and in conserved residues near predicted cofactor binding sites. These differences can be used to distinguish members of these two groups of enzymes and may be relevant to the differences in ferredoxin-dependence and ability to mediate electron-bifurcation. These results show that two phylogenetically distinct syntrophic fatty acid-oxidizing bacteria, Syntrophomonas wolfei a member of the phylum Firmicutes, and S. aciditrophicus, a member of the class Deltaproteobacteria, possess functionally similar [FeFe]-hydrogenases that produce hydrogen from NADH during syntrophic fatty acid oxidation without the involvement of reduced ferredoxin. The reliance on a non-bifurcating NADH-dependent [FeFe]-hydrogenases may explain the obligate requirement that many syntrophic metabolizers have for a hydrogen-using partner microorganism when grown on fatty, aromatic and alicyclic acids.
Highlights
Benzoate and its activated form, benzoyl-CoA, are key intermediates in the microbial degradation of organic matter in anaerobic environments (Harwood et al, 1998; McInerney et al, 2008, 2009)
The specific activity for methyl viologen reduction with hydrogen for multimeric [FeFe]-hydrogenases purified from native organisms were: 70 U · mg−1 for T. maritima HydABC (Schut and Adams, 2009), 760 U · mg−1 for A. woodii HydABCD (Schuchmann and Muller, 2012), 181 U · mg−1 for M. thermoacetica HydABC (Wang et al, 2013c) and 18000 U · mg−1 for C. autoethanogenum FdhA/HytABCDE (Wang et al, 2013a)
The beta subunits of the S. wolfei and S. aciditrophicus [FeFe]hydrogenases share features with the beta subunits of other non-BF NADH-dependent enzymes and these are distinct from the beta subunits of BF [FeFe]-hydrogenases
Summary
Benzoate and its activated form, benzoyl-CoA, are key intermediates in the microbial degradation of organic matter in anaerobic environments (Harwood et al, 1998; McInerney et al, 2008, 2009). Benzoate and other aromatic compounds are degraded to acetate, formate, CO2, and H2 by syntrophic metabolizers. The microbial conversion of benzoate and other aromatic and fatty acids to acetate, formate, CO2, and H2 is thermodynamically unfavorable unless formate and H2 are kept at low levels. This requires the presence of formate- and H2-using microorganisms such as methanogens or sulfate reducers. Syntrophus aciditrophicus is a metabolic specialist capable of oxidizing benzoate, alicyclic acids such as cyclohexane-1-carboxylate, and fatty acids when paired with a hydrogen- or formate-consuming partner organism such as a methanogen or sulfate reducer (Jackson et al, 1999; Elshahed and McInerney, 2001). S. aciditrophicus has electrically conductive pili that allow syntrophic growth by direct electron transfer (Walker et al, 2020)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
