Activation of (R)-2-hydroxyglutaryl-CoA Dehydratase from Acidaminococcus fermentans

Abstract

(R)-2-Hydroxyglutaryl-CoA dehydratase (HgdAB) from Acidaminococcus fermentans catalyses the reversible dehydration of its substrate to glutaconyl-CoA. The enzyme has to be activated by ATP, MgCl2, and Ti(III)citrate by an activator protein (HgdC) that is present in the organism at very low concentrations. Cell-free extracts of a recombinant Escherichia coli strain, in which hgdC was expressed, contained the activator with a specific activity of up to 45 U'/mg protein (1 U' is the amount of activator required to generate 1 U dehydratase activity under standard assay conditions). The recombinant protein was purified 44-fold to a specific activity of 2000 U'/mg. It is a homodimer (gamma 2, 54 kDa) and contains 4 mol non-heme iron and 3 mol inorganic sulfur. Under air, the activator has a half-life of seconds and even under strict anaerobic conditions it is very unstable. The amino acid sequence of the activator shows similarities to the ATP-binding motifs of several kinases. The dehydratase component was purified from its natural source revealing a heterodimer (alpha beta, 100 kDa) that contains 4 mol non-heme iron, 4 mol inorganic sulfur, 0.3 mol riboflavin, and 1 mol FMN. A mechanism is proposed in which an iron-sulfur cluster or a flavin donates one electron to the thiolester of the substrate (R)-2-hydroxyglutaryl-CoA. The resulting ketyl may eliminate the adjacent hydroxyl group yielding an enoxy radical from which the beta-hydrogen is abstracted as a proton leading to the ketyl of glutaconyl-CoA. In the final step, the latter is oxidized to the product, whereby the reduced enzyme is regenerated. It is suggested that during the activation step, the electron of this cycle is fed into the enzyme by Ti(III)citrate and energized by hydrolysis of ATP; both functions are apparently catalysed by the activator. The enzyme remains in this activated state for several turnovers, which may explain the requirement of only catalytic amounts of ATP and substoichiometric amounts of activator (dehydratase/activator ratio approximately 200:1). The oxidants 4-nitrobenzoate, 2-nitrophenol, 3-nitrophenol, 4-nitrophenol, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone or chloramphenicol (all at concentrations greater than or equal to 1 microM) may trap this electron resulting in a reversible, transient inactivation of the dehydratase.

The iron–sulfur clusters in 2‐hydroxyglutaryl‐CoA dehydratase from Acidaminococcus fermentans

“…Incubation with ferricyanide (10‐fold excess) led to denaturation of the protein. Exposure to air for 10 min had almost no effect on the spectroscopic behaviour nor on the activity of component D which clearly showed that it is by far not as oxygen sensitive as described earlier [5]. Incubation under oxic conditions for 24 h at 4 °C caused ≈ 30% loss of activity.…”

“…With a new purification method it was possible to purify 50 mg component D from 30 g wet cells of A. fermentans . Notably, the so isolated protein had a high specific activity of 9 U·mg −1 (15 s −1 ) compared to 5–6 U·mg −1 obtained previously [5]. Analysis of the enzyme gave 4.5 ± 0.3 nonheme iron and 3.5 ± 0.4 acid labile sulfur per heterodimer indicating the presence of a [4Fe−4S] cluster in component D. Upon heating of component D (2 mg·mL −1 , pH 7.0) at 75 °C for 20 min the protein precipitated and released 1.0 ± 0.1 flavin per heterodimer into the supernatant as determined by its absorbancies at 450 nm (ε = 11.3 m m −1 ·cm −1 ) and 370 nm (ε = 10.7 m m −1 ·cm −1 ) [32].…”

“…Under air component D has an apparent half‐life of about an hour, whereas component A loses its activity within seconds. The further characterization of components A and D was hampered by inadequate purification procedures and low stability [4,5]. A mechanism involving ketyl radical anions as reactive intermediates has been postulated for this unusual reaction, in which H 2 O is added to the double bond of glutaconyl‐CoA in an orientation opposite to that catalysed by enoyl‐CoA hydratase of the β‐oxidation pathway of fatty acids.…”

Propionate oxidation in Escherichia coli : evidence for operation of a methylcitrate cycle in bacteria

“…al. 1993;Müller and Buckel 1995) has sequence similarity to an open reading frame from E. coli designated "yijL" at 97 minutes on the chromosome (Burland et al 1995;Buckel 1996). Since 2-hydroxyglutarylCoA dehydratase is closely related to lactoyl-CoA dehydratase, we assumed that in E. coli, propionyl-CoA might indeed be degraded via the α-hydration of acryloyl-CoA.…”

The sodium ion translocating glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans: cloning and function of the genes forming a second operon

“…The formation of ( R )‐2‐hydroxyglutaryl‐CoA by acetyl‐CoA is mediated by glutaconate CoA‐transferase (Buckel et al ., 1981; Jacob et al ., 1997). The subsequent unusual dehydration of ( R )‐2‐hydroxyglutaryl‐CoA to glutaconyl‐CoA is catalysed by an flavin mononucleotide (FMN) and [4Fe–4S] cluster‐containing and ATP‐requiring two‐enzyme system (Buckel, 1980; Müller and Buckel, 1995). Finally, the decarboxylation of glutaconyl‐CoA completes this special pathway leading to the more general intermediate crotonyl‐CoA (eqn 1), which disproportionates to acetate, butyrate and molecular hydrogen (Härtel and Buckel, 1996b).…”