Crystal Structures of Two Bacterial 3-Hydroxy-3-methylglutaryl-CoA Lyases Suggest a Common Catalytic Mechanism among a Family of TIM Barrel Metalloenzymes Cleaving Carbon-Carbon Bonds

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HMG-CoA lyase (HMGCL 4 ; EC 4.1.3.4) catalyzes a cationdependent cleavage of substrate into acetyl-CoA and acetoacetate (Scheme 1) (1). This reaction is a key step in ketogenesis, the products of which support anaplerotic metabolism in bacteria (2) and energy production in nonhepatic animal tissues (3). Ketogenesis is particularly important to human metabolism during the prenatal period and during fasting or starvation. In accordance with these physiological roles, it is not surprising that gene knock-out in mice results in embryonic lethality (4). The physiological importance of the enzyme in humans is underscored by the observation that mutations that diminish HMGCL activity correlate with inherited metabolic disease that can be lethal if uncontrolled (5).A variety of human mutations, including many point mutations in protein-coding exons of the gene, have been documented (6). A computational modeling approach was used to explain the molecular basis for some mutations linked to inherited disease (7). This led to the prediction that HMGCL adopts a ␤/␣-barrel fold and a proposal that the acyl-S-pantetheine moiety of the bound substrate passes through the barrel lumen. Initial structural work on human HMGCL liganded to cation and hydroxyglutarate (8) demonstrated that the folding prediction was reasonable but that the substrate binding proposal was unlikely to be correct. The positions of bound cation and hydroxyglutarate (from hydrolyzed hydroxyglutaryl-CoA) indicated the catalytic site to be positioned at the C-terminal end of the barrel, but the absence of a full acyl-CoA molecule in the experimentally determined structure limited detailed insight into the substrate-binding site.To more fully address questions regarding the conformation of bound substrate, activator cation liganding, details concerning reaction chemistry and specificity, as well as the molecular basis for certain inherited HMGCL deficiencies, new structural information on enzyme bound to an intact acyl-CoA molecule is required. To remedy this need, complexes of the WT enzyme with the competitive inhibitor 3-hydroxyglutaryl-CoA and also of catalytically deficient R41M enzyme with the authentic substrate HMG-CoA have been supplemented with the activator cation Mg 2ϩ , and crystallization of the desired ternary complexes has been accomplished. Diffraction quality crystals have been produced, supporting three-dimensional structural determinations for ternary complexes of enzyme, cation, and either the acyl-CoA substrate or inhibitory analog. These findings are

Section: Interpretation Of Functional Observations In the Context Of mentioning

confidence: 99%

Functional Insights into Human HMG-CoA Lyase from Structures of Acyl-CoA-containing Ternary Complexes

Runquist

Montgomery

et al. 2010

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“…Purification and Crystal Structure Determination of Apo-T. maritima RimO-Selenomethionine-labeled RimO was purified according to a published procedure (18). Crystals were grown at room temperature using 2 ϩ 2 l of microbatch reactions under paraffin oil.…”

Section: Expression and Purification Of T Maritima Rimo-proteinmentioning

confidence: 99%

Post-translational Modification of Ribosomal Proteins

Arragain

García-Serres

Blondin

et al. 2010

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Ribosomes are large ribonucleoprotein complexes that catalyze the peptidyltransferase reaction in protein synthesis and are thus responsible for the translation of transcripts encoded in the cellular genome. Detailed analyses of eukaryote and prokaryote ribosomes by peptide mass spectrometry provide insights into the composition of ribosomal proteins and show a high degree of posttranslational modifications (1). These modifications are believed to extend molecular structures beyond the limits imposed by the 20 genetically encoded amino acids (2). For example, the Escherichia coli ribosomal protein S12 is shown to be post-translationally modified through 3-methylthiolation of the Asp-89 3 residue (Scheme 1A), a modification believed to improve translational accuracy (3, 4). Recently, the yliG gene (later named rimO for ribosomal modification O) has been shown to be responsible for this reaction in vivo (5). The protein encoded by this gene, RimO, contains in its central part the highly conserved cysteine triad Cys-XXX-Cys-XX-Cys, which is the hallmark of the radical AdoMet 4 superfamily (Scheme 1B) (6).Radical AdoMet enzymes share a common mechanism that utilizes a 2ϩ/1ϩ cluster chelated by the three cysteines of the triad and by AdoMet to initiate, under reducing conditions, a radical reaction mediated by a 5Ј-deoxyadenosyl radical arising from the reductive cleavage of the bound AdoMet (7,8). This radical abstracts a hydrogen atom from a properly positioned substrate creating a substrate-based carbon radical. In the formation of 3-methylthioaspartate at Asp-89 of the S12 protein (ms-D89-S12), this radical is supposed to be located on the C3 of Asp-89, and then the site becomes successively thiolated and methylated (9).Several other radical AdoMet enzymes besides RimO catalyze the thiolation of substrates, including a tRNA-methylthio-

“…A number of active site residues, including divalent cation ligands, have been proposed on the basis of site-directed mutagenesis and characterization of mutants by kinetic and electron spin resonance studies. His 235 (9), Asp 42 , and Glu 72 (10) have been proposed to support divalent cation binding to protein. Asp 42 (10), His 233 (6), and Cys 266 (11) are important for catalytic efficiency.…”

mentioning

confidence: 99%

“…His 235 (9), Asp 42 , and Glu 72 (10) have been proposed to support divalent cation binding to protein. Asp 42 (10), His 233 (6), and Cys 266 (11) are important for catalytic efficiency. For D42A, V max is diminished by 1.3 ϫ 10 5 -fold compared with wild type enzyme.…”

mentioning

confidence: 99%

Crystal Structure of Human 3-Hydroxy-3-methylglutaryl-CoA Lyase

Runquist

Forouhar

et al. 2006

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3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is a key enzyme in the ketogenic pathway that supplies metabolic fuel to extrahepatic tissues. Enzyme deficiency may be due to a variety of human mutations and can be fatal. Diminished activity has been explained based on analyses of recombinant human mutant proteins or, more recently, in the context of structural models for the enzyme. We report the experimental determination of a crystal structure at 2.1 Å resolution of the recombinant human mitochondrial HMG-CoA lyase containing a bound activator cation and the dicarboxylic acid 3-hydroxyglutarate. The enzyme adopts a (␤␣) 8 barrel fold, and the N-terminal barrel end is occluded. The structure of a physiologically relevant dimer suggests that substrate access to the active site involves binding across the cavity located at the C-terminal end of the barrel. An alternative hypothesis that involves substrate insertion through a pore proposed to extend through the barrel is not compatible with the observed structure. The activator cation ligands included Asn 235; the latter three residues had been implicated previously as contributing to metal binding or enzyme activity. Arg 41, previously shown to have a major effect on catalytic efficiency, is also located at the active site. In the observed structure, this residue interacts with a carboxyl group of 3-hydroxyglutarate, the hydrolysis product of the competitive inhibitor 3-hydroxyglutaryl-CoA required for crystallization of human enzyme. The structure provides a rationale for the decrease in enzyme activity due to clinical mutations, including H233R, R41Q, D42H, and D204N, that compromise active site function or enzyme stability.3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) 2 lyase (EC 4.1.3.4) catalyzes the cleavage of HMG-CoA into acetyl-CoA and acetoacetate (1). Scheme 1 is a key step in ketogenesis (2), which maintains the energy requirements of nonhepatic tissues especially during fasting or starvation. The reaction is also an important step in leucine catabolism (3). Homologous C-C bond cleavage or condensation reactions are catalyzed by various members of the HMG-CoA lyase family of enzymes, which includes homocitrate synthase, isopropylmalate synthase, and 4-hydroxy-2-ketovalerate aldolase (4).The importance of the ketogenic cycle is underscored in hereditary HMG-CoA lyase deficiency, which can result in hypoketotic hypoglycemia and a marked increase in serum levels of several organic acids. Uncontrolled HMG-CoA lyase deficiency is lethal in about 20% of cases and can result in mental retardation, episodes of seizure, and coma (5). Several mutations in the HMG-CoA lyase gene correlating with deficiency have been identified (5), including the following missense mutations: H233R (6), R41Q, D42E, D42H, D42G (7), and recently E279K (8). These mutations have been studied by using our recombinant human lyase protein expression system, providing a more detailed biochemical explanation for the significant consequences of such mutations.The cleavage of HMG-CoA req...