Abstract
The breakdown of fucose and rhamnose released from plant cell walls by the cellulolytic soil bacterium Clostridium phytofermentans produces toxic aldehyde intermediates. To enable growth on these carbon sources, the pathway for the breakdown of fucose and rhamnose is encapsulated within a bacterial microcompartment (BMC). These proteinaceous organelles sequester the toxic aldehyde intermediates and allow the efficient action of acylating aldehyde dehydrogenase enzymes to produce an acyl-CoA that is ultimately used in substrate-level phosphorylation to produce ATP. Here we analyse the kinetics of the aldehyde dehydrogenase enzyme from the fucose/rhamnose utilisation BMC with different short-chain fatty aldehydes and show that it has activity against substrates with up to six carbon atoms, with optimal activity against propionaldehyde. We have also determined the X-ray crystal structure of this enzyme in complex with CoA and show that the adenine nucleotide of this cofactor is bound in a distinct pocket to the same group in NAD+. This work is the first report of the structure of CoA bound to an aldehyde dehydrogenase enzyme and our crystallographic model provides important insight into the differences within the active site that distinguish the acylating from non-acylating aldehyde dehydrogenase enzymes.
Highlights
Ki n/a as the production of acetaldehyde from the choline utilisation Bacterial microcompartments (BMCs) found in Desulfovibrio desulfuricans[14] and the C. phytofermentans fucose/rhamnose BMC13
The aldehyde dehydrogenase enzymes (AldDH) encoded with BMC loci are invariably accompanied by an alcohol dehydrogenase enzyme[17], the activity of which appears to be necessary to recycle the NADH produced by the activity of the aldehyde dehydrogenase
Cphy1178 enzyme displayed the lowest KM and the highest kcat/KM values for the substrates tested. This data is consistent with the hypothesis that this protein acts as a propionaldehyde dehydrogenase within the C. phytofermentans fucose/rhamnose BMC
Summary
Ki (mM) n/a as the production of acetaldehyde from the choline utilisation BMC found in Desulfovibrio desulfuricans[14] and the C. phytofermentans fucose/rhamnose BMC13. The aldehyde dehydrogenase superfamily are well studied and are active against a range of aldehydes, including the short chain fatty aldehyde products of the lyase enzymes in BMCs19–21 These enzymes have a common architecture, with a Rossman-fold nucleotide-binding domain that positions the NAD(P)+ cofactor required for hydride transfer from the aldehyde substrate[22]; the catalytic domain has a substrate-binding tunnel with a catalytic cysteine residue and glutamic acid residue that acts as a general base in the hydrolysis of the acyl-enzyme intermediate[23]. We have determined the structure of the protein in complex with its cofactors NAD+ and CoA and show that the adenine nucleotides of these co-factors adopt different conformations within the Rossman fold domain
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