Unexpected stereoselective exchange of straight-chain fatty acyl-CoA alpha-protons by human alpha-methylacyl-CoA racemase 1A (P504S).

Alpha-methylacyl-CoA racemase (AMACR; P504S) catalysed exchange of straight-chain fatty acyl-CoA alpha-protons. One alpha-proton was removed in each catalytic cycle, with the pro-S proton preferred. This reaction was most efficient for straight-chain substrates with longer side-chains. 2-Methyldecanoyl-CoA underwent alpha-proton exchange 3x more efficiently (as judged by K(cat)/K(m)) than decanoyl-CoA.

Synthesis and use of isotope-labelled substrates for a mechanistic study on human alpha-methylacyl-CoA racemase 1A (AMACR; P504S).

Alpha-Methylacyl-CoA racemase (AMACR) is an important enzyme for the metabolism of branched-chain lipids and drugs. The enzyme is over-expressed in prostate and other cancers. AMACR 1A, the major splice variant, was purified from recombinant E. coli cells as a His-tag protein. Purified enzyme catalysed chiral inversion of both S- and R-2-methyldecanoyl-CoA, with an equilibrium constant of 1.09 +/- 0.14 (2S/2R). Reactions with (2)H-labelled substrate showed that loss of the alpha-proton was a prerequisite for chiral inversion. Reactions conducted in (2)H(2)O indicated that reprotonation was not stereospecific. These results are the first mechanistic study on any recombinant mammalian alpha-methylacyl-CoA racemase.

An allylic ketyl radical intermediate in clostridial amino-acid fermentation.

The human pathogenic bacterium Clostridium difficile thrives by the fermentation of l-leucine to ammonia, CO(2), 3-methylbutanoate and 4-methylpentanoate under anaerobic conditions. The reductive branch to 4-methylpentanoate proceeds by means of the dehydration of (R)-2-hydroxy-4-methylpentanoyl-CoA to 4-methylpent-2-enoyl-CoA, which is chemically the most demanding step. Ketyl radicals have been proposed to mediate this reaction catalysed by an iron-sulphur-cluster-containing dehydratase, which requires activation by ATP-dependent electron transfer from a second iron-sulphur protein functionally similar to the iron protein of nitrogenase. Here we identify a kinetically competent product-related allylic ketyl radical bound to the enzyme by electron paramagnetic resonance spectroscopy employing isotope-labelled (R)-2-hydroxy-4-methylpentanoyl-CoA species. We also found that the enzyme generated the stabilized pentadienoyl ketyl radical from the substrate analogue 2-hydroxypent-4-enoyl-CoA, supporting the proposed mechanism. Our results imply that also other 2-hydroxyacyl-CoA dehydratases and the related benzoyl-CoA reductases-present in anaerobically living bacteria-employ ketyl radical intermediates. The absence of radical generators such as coenzyme B12, S-adenosylmethionine or oxygen makes these enzymes unprecedented in biochemistry.

Alpha-methylacyl-CoA racemase--an 'obscure' metabolic enzyme takes centre stage.

Branched-chain lipids are important components of the human diet and are used as drug molecules, e.g. ibuprofen. Owing to the presence of methyl groups on their carbon chains, they cannot be metabolized in mitochondria, and instead are processed and degraded in peroxisomes. Several different oxidative degradation pathways for these lipids are known, including alpha-oxidation, beta-oxidation, and omega-oxidation. Dietary branched-chain lipids (especially phytanic acid) have attracted much attention in recent years, due to their link with prostate, breast, colon and other cancers as well as their role in neurological disease. A central role in all the metabolic pathways is played by alpha-methylacyl-CoA racemase (AMACR), which regulates metabolism of these lipids and drugs. AMACR catalyses the chiral inversion of a diverse number of 2-methyl acids (as their CoA esters), and regulates the entry of branched-chain lipids into the peroxisomal and mitochondrial beta-oxidation pathways. This review brings together advances in the different disciplines, and considers new research in both the metabolism of branched-chain lipids and their role in cancer, with particular emphasis on the crucial role played by AMACR. These recent advances enable new preventative and treatment strategies for cancer.

Characterization of (R)-2-hydroxyisocaproate dehydrogenase and a family III coenzyme A transferase involved in reduction of L-leucine to isocaproate by Clostridium difficile.

The strictly anaerobic pathogenic bacterium Clostridium difficile occurs in the human gut and is able to thrive from fermentation of leucine. Thereby the amino acid is both oxidized to isovalerate plus CO(2) and reduced to isocaproate. In the reductive branch of this pathway, the dehydration of (R)-2-hydroxyisocaproyl-coenzyme A (CoA) to (E)-2-isocaprenoyl-CoA is probably catalyzed via radical intermediates. The dehydratase requires activation by an ATP-dependent one-electron transfer (J. Kim, D. Darley, and W. Buckel, FEBS J. 272:550-561, 2005). Prior to the dehydration, a dehydrogenase and a CoA transferase are supposed to be involved in the formation of (R)-2-hydroxyisocaproyl-CoA. Deduced amino acid sequences of ldhA and hadA from the genome of C. difficile showed high identities to d-lactate dehydrogenase and family III CoA transferase, respectively. Both putative genes encoding the dehydrogenase and CoA transferase were cloned and overexpressed in Escherichia coli; the recombinant Strep tag II fusion proteins were purified to homogeneity and characterized. The substrate specificity of the monomeric LdhA (36.5 kDa) indicated that 2-oxoisocaproate (K(m) = 68 muM, k(cat) = 31 s(-1)) and NADH were the native substrates. For the reverse reaction, the enzyme accepted (R)- but not (S)-2-hydroxyisocaproate and therefore was named (R)-2-hydroxyisocaproate dehydrogenase. HadA showed CoA transferase activity with (R)-2-hydroxyisocaproyl-CoA as a donor and isocaproate or (E)-2-isocaprenoate as an acceptor. By site-directed mutagenesis, the conserved D171 was identified as an essential catalytic residue probably involved in the formation of a mixed anhydride with the acyl group of the thioester substrate. However, neither hydroxylamine nor sodium borohydride, both of which are inactivators of the CoA transferase, modified this residue. The dehydrogenase and the CoA transferase fit well into the proposed pathway of leucine reduction to isocaproate.

Molecular and functional analysis of nicotinate catabolism in Eubacterium barkeri.

The anaerobic soil bacterium Eubacterium barkeri catabolizes nicotinate to pyruvate and propionate via a unique fermentation. A full molecular characterization of nicotinate fermentation in this organism was accomplished by the following results: (i) A 23.2-kb DNA segment with a gene cluster encoding all nine enzymes was cloned and sequenced, (ii) two chiral intermediates were discovered, and (iii) three enzymes were found, completing the hitherto unknown part of the pathway. Nicotinate dehydrogenase, a (nonselenocysteine) selenium-containing four-subunit enzyme, is encoded by ndhF (FAD subunit), ndhS (2 x [2Fe-2S] subunit), and by the ndhL/ndhM genes. In contrast to all enzymes of the xanthine dehydrogenase family, the latter two encode a two-subunit molybdopterin protein. The 6-hydroxynicotinate reductase, catalyzing reduction of 6-hydroxynicotinate to 1,4,5,6-tetrahydro-6-oxonicotinate, was purified and shown to contain a covalently bound flavin cofactor, one [2Fe-2S](2+/1+) and two [4Fe-4S](2+/1+) clusters. Enamidase, a bifunctional Fe-Zn enzyme belonging to the amidohydrolase family, mediates hydrolysis of 1,4,5,6-tetrahydro-6-oxonicotinate to ammonia and (S)-2-formylglutarate. NADH-dependent reduction of the latter to (S)-2-(hydroxymethyl)glutarate is catalyzed by a member of the 3-hydroxyisobutyrate/phosphogluconate dehydrogenase family. A [4Fe-4S]-containing serine dehydratase-like enzyme is predicted to form 2-methyleneglutarate. After the action of the coenzyme B(12)-dependent 2-methyleneglutarate mutase and 3-methylitaconate isomerase, an aconitase and isocitrate lyase family pair of enzymes, (2R,3S)-dimethylmalate dehydratase and lyase, completes the pathway. Genes corresponding to the first three enzymes of the E. barkeri nicotinate catabolism were identified in nine Proteobacteria.

2-Hydroxyisocaproyl-CoA dehydratase and its activator from Clostridium difficile.

The hadBC and hadI genes from Clostridium difficile were functionally expressed in Escherichia coli and shown to encode the novel 2-hydroxyisocaproyl-CoA dehydratase HadBC and its activator HadI. The activated enzyme catalyses the dehydration of (R)-2-hydroxyisocaproyl-CoA to isocaprenoyl-CoA in the pathway of leucine fermentation. The extremely oxygen-sensitive homodimeric activator as well as the heterodimeric dehydratase, contain iron and inorganic sulfur; besides varying amounts of zinc, other metal ions, particularly molybdenum, were not detected in the dehydratase. The reduced activator transfers one electron to the dehydratase concomitant with hydrolysis of ATP, a process similar to that observed with the unrelated nitrogenase. The thus activated dehydratase was separated from the activator and ATP; it catalyzed about 10(4) dehydration turnovers until the enzyme became inactive. Adding activator, ATP, MgCl(2), dithionite and dithioerythritol reactivated the enzyme. This is the first demonstration with a 2-hydroxyacyl-CoA dehydratase that the catalytic electron is recycled after each turnover. In agreement with this observation, only substoichiometric amounts of activator (dehydratase/activator = 10 mol/mol) were required to generate full activity.