Publications

• The bifunctional role of LiuE from Pseudomonas aeruginosa, displays additionally HIHG-CoA lyase enzymatic activity.

  Chavez-Aviles M., Diaz-Perez A.L., Campos-Garcia J.

  Pseudomonas aeruginosa is able to utilize leucine/isovalerate and acyclic terpenes as sole carbon sources. Key enzymes which play an important role in these catabolic pathways are 3-hydroxy-3-methylglutaryl-coenzyme A (CoA) lyase (EC 4.1.3.4; HMG-CoA lyase) and the 3-hydroxy-3-isohexenylglutaryl-C ... >> More

  Pseudomonas aeruginosa is able to utilize leucine/isovalerate and acyclic terpenes as sole carbon sources. Key enzymes which play an important role in these catabolic pathways are 3-hydroxy-3-methylglutaryl-coenzyme A (CoA) lyase (EC 4.1.3.4; HMG-CoA lyase) and the 3-hydroxy-3-isohexenylglutaryl-CoA lyase (EC 4.1.2.26; HIHG-CoA lyase), respectively. HMG-CoA lyase is encoded by the liuE gene while the gene for HIHG-CoA lyase remains unidentified. A mutant in the liuE gene was unable to utilize both leucine/isovalerate and acyclic terpenes indicates an involvement of liuE in both catabolic pathways (Chávez-Avilés et al. 2009, FEMS Microbiol Lett 296:117-123). The LiuE protein was purified as a His-tagged recombinant protein and in addition to show HMG-CoA lyase activity (Chávez-Avilés et al. 2009, FEMS Microbiol Lett 296:117-123), also displays HIHG-CoA lyase activity, indicating a bifunctional role in both the leucine/isovalerate and acyclic terpenes catabolic pathways. << Less

  Mol. Biol. Rep. 37:1787-1791(2010) [PubMed] [EuropePMC]

• Avian 3-hydroxy-3-methylglutaryl-CoA lyase: sensitivity of enzyme activity to thiol/disulfide exchange and identification of proximal reactive cysteines.

  Hruz P.W., Miziorko H.M.

  Catalysis by purified avian 3-hydroxy-3-methylglutaryl-CoA lyase is critically dependent on the reduction state of the enzyme, with less than 1% of optimal activity being observed with the air-oxidized enzyme. The enzyme is irreversibly inactivated by sulfhydryl-directed reagents with the rate of ... >> More

  Catalysis by purified avian 3-hydroxy-3-methylglutaryl-CoA lyase is critically dependent on the reduction state of the enzyme, with less than 1% of optimal activity being observed with the air-oxidized enzyme. The enzyme is irreversibly inactivated by sulfhydryl-directed reagents with the rate of this inactivation being highly dependent upon the redox state of a critical cysteine. Methylation of reduced avian lyase with 1 mM 4-methylnitrobenzene sulfonate results in rapid inactivation of the enzyme with a k(inact) of 0.178 min-1. The oxidized enzyme is inactivated at a sixfold slower rate (k(inact) = 0.028 min-1). Inactivation of the enzyme with the reactive substrate analog 2-butynoyl-CoA shows a similar dependence upon the enzyme's redox state, with a sevenfold difference in k(inact) observed with oxidized vs. reduced forms of the enzyme. Chemical cross-linking of the reduced enzyme with stoichiometric amounts of the bifunctional reagents 1,3-dibromo-2-propanone (DBP) or N,N'-ortho-phenylene-dimaleimide (PDM) coincides with rapid inactivation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of enzyme treated with bifunctional reagent reveals a band of twice the molecular weight of the lyase monomer, indicating that an intersubunit cross-link has been formed. Differential labeling of native and cross-linked protein with [1-14C]iodoacetate has identified as the primary cross-linking target a cysteine within the sequence VSQAACR, which maps at the carboxy-terminus of the cDNA-deduced sequence of the avian enzyme (Mitchell, G.A., et al., 1991, Am. J. Hum. Genet. 49, 101). In contrast, bacterial HMG-CoA lyase, which contains no corresponding cysteine, is not cross-linked by comparable treatment with bifunctional reagent. These results provide evidence for a potential regulatory mechanism for the eukaryotic enzyme via thiol/disulfide exchange and identify a cysteinyl residue with the reactivity and juxtaposition required for participation in disulfide formation. << Less

  Protein Sci 1:1144-1153(1992) [PubMed] [EuropePMC]

• 3-hydroxy-3-methylglutaryl coenzyme A lyase (HL). Cloning of human and chicken liver HL cDNAs and characterization of a mutation causing human HL deficiency.

  Mitchell G.A., Robert M.-F., Hruz P.W., Wang S., Fontaine G., Behnke C.E., Mende-Mueller L.M., Schappert K., Lee C., Gibson K.M., Miziorko H.M. , et al.

  3-Hydroxy-3-methylglutaryl coenzyme A lyase (HL) catalyzes the final step of ketogenesis, an important pathway of mammalian energy metabolism. HL deficiency is an autosomal recessive inborn error in man leading to episodes of hypoglycemia and coma. Using the N-terminal peptide sequence of purified ... >> More

  3-Hydroxy-3-methylglutaryl coenzyme A lyase (HL) catalyzes the final step of ketogenesis, an important pathway of mammalian energy metabolism. HL deficiency is an autosomal recessive inborn error in man leading to episodes of hypoglycemia and coma. Using the N-terminal peptide sequence of purified chicken liver HL, we designed degenerate sequence primers and amplified an 89-base pair (bp) chicken liver HL cDNA fragment. Longer cDNA clones for chicken (1384 bp) and human (1575 bp) HL were obtained by library screening. The peptide sequence predicted from the chicken clone contains two peptides from purified chicken HL. Mature human and chicken HL are 298-residue peptides. The sequence of the human clone predicts a 27-residue mitochondrial leader and a 31.6-kDa mature HL peptide. Human fibroblast and liver RNA contain a single 1.7-kilobase HL message. Two Acadian French-Canadian siblings with HL deficiency were homozygous for a 2-base pair deletion within the Ser-69 codon (S69fs(-2)), predicted to result in a truncated nonfunctional HL peptide lacking a complete active site. S69fs(-2) was not present in 12 other HL-deficient patients of 10 other ethnic origins, showing that HL deficiency is genetically heterogeneous. << Less

  J. Biol. Chem. 268:4376-4381(1993) [PubMed] [EuropePMC]

• 3-Hydroxy-3-methylglutaryl-CoA lyase: expression and isolation of the recombinant human enzyme and investigation of a mechanism for regulation of enzyme activity.

  Roberts J.R., Narasimhan C., Hruz P.W., Mitchell G.A., Miziorko H.M.

  cDNA encoding the mature form of human hydroxy-methylglutaryl-CoA (HMG-CoA) lyase, a mitochondrial matrix protein, has been used to prepare expression plasmids appropriate for production of this protein in Escherichia coli. Using a T7 RNA polymerase-based pET system, HMG-CoA lyase was overexpresse ... >> More

  cDNA encoding the mature form of human hydroxy-methylglutaryl-CoA (HMG-CoA) lyase, a mitochondrial matrix protein, has been used to prepare expression plasmids appropriate for production of this protein in Escherichia coli. Using a T7 RNA polymerase-based pET system, HMG-CoA lyase was overexpressed but largely recovered in an insoluble, catalytically inactive form. In contrast, an expression plasmid (pTrcHL-1), derived from pTrc99a, supported production of soluble, active enzyme. A synthetic oligonucleotide cassette was employed to produce an enzyme variant in which cysteine was replaced by serine at position 323. Both wild-type and C323S HMG-CoA lyases were isolated in homogeneous form and characterized. The function of Cys-323 in influencing catalytic activity in vitro has been investigated by comparing the response of wild-type and C323S lyases to oxidation and reduction. Additionally, the consequences of treatment of these enzymes with the sulfhydryl-directed bifunctional reagent, o-phenylenedimaleimide have been determined. The results support the hypothesis that a thiol/disulfide exchange mechanism affects enzyme activity in vitro and indicate that Cys-323 residues on adjacent subunits of the homodimeric native enzyme are suitably positioned to form an intersubunit cross-link upon oxidative inactivation and disulfide formation. << Less

  J Biol Chem 269:17841-17846(1994) [PubMed] [EuropePMC]

• Identification and characterization of an extramitochondrial human 3-Hydroxy-3-methylglutaryl-CoA lyase.

  Montgomery C., Pei Z., Watkins P.A., Miziorko H.M.

  3-Hydroxy-3-methylglutaryl-CoA lyase-like protein (HMGCLL1) has been annotated in the Mammalian Genome Collection as a previously unidentified human HMG-CoA lyase (HMGCL). To test the validity of this annotation and evaluate the physiological role of the protein, plasmids were constructed for prot ... >> More

  3-Hydroxy-3-methylglutaryl-CoA lyase-like protein (HMGCLL1) has been annotated in the Mammalian Genome Collection as a previously unidentified human HMG-CoA lyase (HMGCL). To test the validity of this annotation and evaluate the physiological role of the protein, plasmids were constructed for protein expression in Escherichia coli and Pichia pastoris. Protein expression in E. coli produced insoluble material. In contrast, active HMGCLL1 could be recovered upon expression in P. pastoris. Antibodies were prepared against a unique peptide sequence found in the N terminus of the protein. In immunodetection experiments, the antibodies discriminated between HMGCLL1 and mitochondrial HMGCL. Purified enzyme was characterized and demonstrated to cleave HMG-CoA to acetoacetate and acetyl-CoA with catalytic and affinity properties comparable with human mitochondrial HMGCL. The deduced HMGCLL1 sequence contains an N-terminal myristoylation motif; the putative modification site was eliminated by construction of a G2A HMGCLL1. Modification of both proteins was attempted using human N-myristoyltransferase and [(3)H]myristoyl-CoA. Wild-type protein was clearly modified, whereas G2A protein was not labeled. Myristoylation of HMGCLL1 affects its cellular localization. Upon transfection of appropriate expression plasmids into COS1 cells, immunofluorescence detection indicates that G2A HMGCLL1 exhibits a diffuse pattern, suggesting a cytosolic location. In contrast, wild-type HMGCLL1 exhibits a punctate as well as a perinuclear immunostaining pattern, indicating myristoylation dependent association with nonmitochondrial membrane compartments. In control experiments with the HMGCL expression plasmid, protein is localized in the mitochondria, as anticipated. The available results for COS1 cell expression, as well as endogenous expression in U87 cells, indicate that HMGCLL1 is an extramitochondrial hydroxymethylglutaryl-CoA lyase. << Less

  J. Biol. Chem. 287:33227-33236(2012) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization of a novel HMG-CoA Lyase enzyme with a dual location in endoplasmic reticulum and cytosol.

  Arnedo M., Menao S., Puisac B., Teresa-Rodrigo M.E., Gil-Rodriguez M.C., Lopez-Vinas E., Gomez-Puertas P., Casals N., Casale C.H., Hegardt F.G., Pie J.

  A novel lyase activity enzyme is characterized for the first time: HMG-CoA lyase-like1 (er-cHL), which is a close homolog of mitochondrial HMG-CoA lyase (mHL). Initial data show that there are nine mature transcripts for the novel gene HMGCLL1, although none of them has all its exons. The most abu ... >> More

  A novel lyase activity enzyme is characterized for the first time: HMG-CoA lyase-like1 (er-cHL), which is a close homolog of mitochondrial HMG-CoA lyase (mHL). Initial data show that there are nine mature transcripts for the novel gene HMGCLL1, although none of them has all its exons. The most abundant transcript is called "variant b," and it lacks exons 2 and 3. Moreover, a three-dimensional model of the novel enzyme is proposed. Colocalization studies show a dual location of the er-cHL in the endoplasmic reticulum (ER) and cytosol, but not in mitochondria or peroxisomes. Furthermore, the dissociation experiment suggests that it is a nonendoplasmic reticulum integral membrane protein. The kinetic parameters of er-cHL indicate that it has a lower V(max) and a higher substrate affinity than mHL. Protein expression and lyase activity were found in several tissues, and were particularly strong in lung and kidney. The occurrence of er-cHL in brain is surprising, as mHL has not been found there. Although mHL activity is clearly associated with energy metabolism, the results suggest that er-cHL is more closely related to another metabolic function, mostly at the pulmonary and brain level. << Less

  J. Lipid Res. 53:2046-2056(2012) [PubMed] [EuropePMC]

• Evaluation of 3-hydroxy-3-methylglutaryl-coenzyme A lyase arginine-41 as a catalytic residue: use of acetyldithio-coenzyme A to monitor product enolization.

  Tuinstra R.L., Wang C.-Z., Mitchell G.A., Miziorko H.M.

  3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase catalyzes the divalent cation-dependent cleavage of HMG-CoA to produce acetyl-CoA and acetoacetate. Arginine-41 is an invariant residue in HMG-CoA lyases. Mutation of this residue (R41Q) correlates with human HMG-CoA lyase deficiency. To evalua ... >> More

  3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase catalyzes the divalent cation-dependent cleavage of HMG-CoA to produce acetyl-CoA and acetoacetate. Arginine-41 is an invariant residue in HMG-CoA lyases. Mutation of this residue (R41Q) correlates with human HMG-CoA lyase deficiency. To evaluate the functional importance of arginine-41, R41Q and R41M recombinant mutant human HMG-CoA lyase proteins have been constructed, expressed, and purified. These mutant proteins retain structural integrity based on Mn(2+) binding and affinity labeling stoichiometry. R41Q exhibits a 10(5)-fold decrease in V(max); R41M activity is >or=10-fold lower than the activity of R41Q. Acetyldithio-CoA, an analogue of the reaction product, acetyl-CoA, has been employed to test the function of arginine-41, as well as other residues (e.g., aspartate-42 and histidine-233) implicated in catalysis. Acetyldithio-CoA supports enzyme-catalyzed exchange of the methyl protons of the acetyl group with solvent; exchange is dependent on the presence of Mg(2+) and acetoacetate. In comparison with wild-type human enzyme, D42A and H233A mutant enzymes exhibit 4-fold and 10-fold decreases, respectively, in the proton exchange rate. In contrast, R41Q and R41M mutants do not catalyze any substantial enzyme-dependent proton exchange. These results suggest a role for arginine-41 in deprotonation or enolization of acetyldithio-CoA and implicate this residue in the HMG-CoA cleavage reaction chemistry that leads to acetyl-CoA product formation. Assignment of arginine-41 as an active site residue is also supported by a homology model for HMG-CoA lyase based on the structure of 4-hydroxy-2-ketovalerate aldolase. This model suggests the proximity of arginine-41 to other amino acids (aspartate-42, glutamate-72, histidine-235) implicated as active site residues based on their function as ligands to the activator cation. << Less

  Biochemistry 43:5287-5295(2004) [PubMed] [EuropePMC]