Acryloyl‐CoA reductase from Clostridium propionicum

Abstract

Acryloyl-CoA reductase from Clostridium propionicum catalyses the irreversible NADH-dependent formation of propionyl-CoA from acryloyl-CoA. Purification yielded a heterohexadecameric yellow-greenish enzyme complex [(a 2 bc) 4 ; molecular mass 600 ± 50 kDa] composed of a propionyl-CoA dehydrogenase (a 2 , 2 · 40 kDa) and an electron-transferring flavoprotein (ETF; b, 38 kDa; c, 29 kDa). A flavin content (90% FAD and 10% FMN) of 2.4 mol per a 2 bc subcomplex (149 kDa) was determined. A substrate alternative to acryloyl-CoA (K m ¼ 2 ± 1 lM; k cat ¼ 4.5 s )1 at 100 lM NADH) is 3-buten-2-one (methyl vinyl ketone; K m ¼ 1800 lM; k cat ¼ 29 s )1 at 300 lM NADH). The enzyme complex exhibits acyl-CoA dehydrogenase activity with propionyl-CoA (K m ¼ 50 lM;as electron donor and 200 lM ferricenium hexafluorophosphate as acceptor. The enzyme also catalysed the oxidation of NADH by iodonitrosotetrazolium chloride (diaphorase activity) or by air, which led to the formation of H 2 O 2 (NADH oxidase activity). The N-terminus of the dimeric propionyl-CoA dehydrogenase subunit is similar to those of butyryl-CoA dehydrogenases from several clostridia and related anaerobes (up to 55% sequence identity). The N-termini of the b and c subunits share 40% and 35% sequence identities with those of the A and B subunits of the ETF from Megasphaera elsdenii, respectively, and up to 60% with those of putative ETFs from other anaerobes. Acryloyl-CoA reductase from C. propionicum has been characterized as a soluble enzyme, with kinetic properties perfectly adapted to the requirements of the organism. The enzyme appears not to be involved in anaerobic respiration with NADH or reduced ferredoxin as electron donors. There is no relationship to the trans-2-enoyl-CoA reductases from various organisms or the recently described acryloylCoA reductase activity of propionyl-CoA synthase from Chloroflexus aurantiacus.Keywords: acryloyl-CoA reductase; Clostridium propionicum; electron-transferring flavoprotein; NADH oxidase; propionyl-CoA dehydrogenase.Several anaerobic bacteria belonging to the phyla Firmicutes (Gram-positives, phylum BXIII) and Fusobacteria (phylum BXXI) [1] have the unique ability to conserve energy by fermentation of amino acids. Clostridium propionicum (BXIII) degrades alanine (Eqn 1) and serine to carbon dioxide, ammonia, acetate and propionate [2], whereas with threonine as substrate the short-chain fatty acids formed are propionate and butyrate [3]; for reviews see [4,5].The pathway of alanine fermentation proceeds via pyruvate, which is oxidized to acetate and reduced to propionate (Fig. 1) [6,7]. Pyruvate is produced by amino transfer from alanine to 2-oxoglutarate, which is regenerated by the action of an NAD + -dependent glutamate dehydrogenase, whereby ammonia and NADH are formed.

Rhodobacter sphaeroides Uses a Reductive Route via Propionyl Coenzyme A To Assimilate 3-Hydroxypropionate

“…The absence of an acuI-like gene in other DMSP-utilizing organisms, on the other hand, does not necessarily rule out the assimilation of carbon by a reductive route. In principle, enzymes with very limited sequence identity to AcuI or members of different enzyme classes might catalyze the reduction of acrylyl-CoA to propionyl-CoA (2,22,42).…”

The compositional and evolutionary logic of metabolism

“…Even the distinctive step to crotonyl-CoA in the 4HB pathway creates an aconate-type intermediate, and the enzyme responsible has high homology to the acrolyl-CoA synthetase [139,140], whose output (acrolyl-CoA) follows the standard pattern. Only the position of the double bond breaks the strict pattern in crotonyl-CoA, and the abstraction of the un-activated proton required to produce this bond requires the unique ketyl-radical intermediate [141].…”

Stabilisation of Methylene Radicals by Cob(II)alamin in Coenzyme B12 Dependent Mutases

“…Reversible dehydration to acryloyl-CoA ensues, followed by an irreversible NADH-dependent reduction to propionylCoA. [34] Notably the removal of a hydrogen atom from the methyl group of lactyl-CoA (pK > 40), during the dehydration to acryloyl-CoA, also requires radical chemistry. It has been proposed that the reaction proceeds via resonance-stabilised ketyl radicals (i.e., radical anions) rather than a much less stable methylene radical.…”

“…It has been proposed that the reaction proceeds via resonance-stabilised ketyl radicals (i.e., radical anions) rather than a much less stable methylene radical. [35,36] The pathway via acryloyl-CoA has three drawbacks: it is irreversible in the direction of propionate formation, [34] acryloyl-CoA is toxic [37] and lactyl-CoA dehydratase is an extremely oxygen-sensitive enzyme. [38] Hence, aerobic bacteria such as enterobacteria and pseudomonads, as well as in fungi and probably plants, all of which lack coenzyme B 12 , are forced to oxidise propionate by a different pathway.…”