Substrate-Induced Radical Formation in 4-Hydroxybutyryl Coenzyme A Dehydratase from Clostridium aminobutyricum

Abstract

2؉ cluster prior to hydration. We describe an active recombinant 4HBD and variants produced in Escherichia coli. The variants of the cluster ligands (H292C [histidine at position 292 is replaced by cysteine], H292E, C99A, C103A, and C299A) had no measurable dehydratase activity and were composed of monomers, dimers, and tetramers. Variants of other potential catalytic residues were composed only of tetramers and exhibited either no measurable (E257Q, E455Q, and Y296W) hydratase activity or <1% (Y296F and T190V) dehydratase activity. The E455Q variant but not the Y296F or E257Q variant displayed the same spectral changes as the wild-type enzyme after the addition of crotonyl-CoA but at a much lower rate. The results suggest that upon the addition of a substrate, Y296 is deprotonated by E455 and reduces FAD to FADH·, aided by protonation from E257 via T190. In contrast to FADH·, the tyrosyl radical could not be detected by EPR spectroscopy. FADH· appears to initiate the radical dehydration via an allylic ketyl radical that was proposed 19 years ago. The mode of radical generation in 4HBD is without precedent in anaerobic radical chemistry. It differs largely from that in enzymes, which use coenzyme B 12 , S-adenosylmethionine, ATP-driven electron transfer, or flavin-based electron bifurcation for this purpose. 4-Hydroxybutyryl-coenzyme A (CoA) dehydratase (4HBD) catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA and the irreversible ⌬-isomerization of vinylacetyl-CoA to crotonyl-CoA (Fig. 1). The enzyme was discovered in the fermentation of 4-aminobutyrate (␥-aminobutyrate [GABA]) to ammonia, acetate, and butyrate (Fig. 2) by Clostridium aminobutyricum (1, 2), attributed to cluster XI of the clostridia (3, 4). In this pathway, 4-aminobutyrate is transaminated with 2-oxoglutarate to yield glutamate and succinic semialdehyde (4-oxobutanoate), which is reduced to 4-hydroxybutyrate. Activation to the CoA thioester and dehydration affords crotonyl-CoA, which disproportionates to butyrate and acetate. Energy is conserved via substrate level phosphorylation via acetylphosphate and via electron bifurcation at the electron-transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (5, 6). The reduced ferredoxin obtained thereby recycles NADH, mediated by NAD-ferredoxin oxidoreductase, also called Rnf, which generates an electrochemical Na ϩ gradient for ATP synthesis (7,8). With one additional dehydrogenase, this pathway is used by Clostridium kluyveri to reduce succinyl-CoA to butyrate (Fig. 2) (9). Autotrophic CO 2 -fixing Crenarchaeota synthesize succinyl-CoA by reductive carboxylations of acetyl-CoA. The pathway depicted in Fig. 2 recycles the carrier molecule acetyl-CoA and forms a second one for biosynthesis (10, 11). The key enzyme of all these conversions is 4HBD, which links lipid and carbohydrate metabolisms. In this work, we study the enzyme in more detail and propose a plausible mechanism of action.4HBD from C. aminobutyricum is a homotetrameric enzyme (4ϫ 56 kDa) containing one 2ϩ...

Two Pathways for Glutamate Biosynthesis in the Syntrophic Bacterium Syntrophus aciditrophicus

“…In this pathway, 4-aminobutyrate (␥-aminobutyrate [GABA]), formed by decarboxylation of glutamate in other organisms (32), is transaminated to succinate semialdehyde followed by reduction to 4-hydroxybutyrate. CoA transfer yields 4-hydroxybutyryl-CoA that is reversibly dehydrated to crotonyl-CoA mediated by a flavin adenine dinucleotide (FAD) and [4Fe-4S]-containing dehydratase (24,33). Consecutive disproportionation of crotonyl-CoA leads to acetate and butyrate.…”

“…A detailed description of the assays with 4-hydroxybutyrate or with vinyl acetate as the substrate has been published recently (24). Because several other enzymes catalyze the ⌬-isomerization of vinyl acetyl-CoA to crotonyl-CoA, in the assays with cell extracts described here, vinyl acetate has been replaced by 4-hydroxybutyrate.…”

Anaerobic Radical Enzymes for Biotechnology

“…For these reactions, a radical is proposed to enact an umpolung reaction via an intermediate ketyl radical, which allows subsequent deprotonation and dehydration. The deprotonation in 1,2‐ and 1,4‐dehydratases is typically initiated by a single electron reduction via either an archerase or an FAD‐dependent oxidation , , respectively, both mediated with an Fe 4 S 4 cluster. The archerase activators of 2‐hydroxyacyl‐CoA dehydratases are so named because they evoke the image of an archer in their action, shooting an electron into the dehydratase, driven by ATP hydrolysis.…”

Structural adaptation of oxygen tolerance in 4-hydroxybutyrl-CoA dehydratase, a key enzyme of archaeal carbon fixation

“…The enzyme requires FAD as a cofactor, as well as a [4Fe-4S] cluster in the active site 15 . To date, structural and biochemical characterization of 4HBD has primarily focused on previously-known anaerobic versions, specifically in C. aminobutyricum [14][15][16][17] . More recently, a comparative functional characterization of 4HBD from anaerobic C. aminobutyricum and aerobic N. maritimus confirmed the stark difference in their oxygen sensitivity, ascribing it to a key evolutionary adaptation of AOA to an oxic environment 13 .…”