Publications

• 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.

• Effects of L-histidine and its structural analogues on human N-myristoyltransferase activity and importance of EEVEH amino acid sequence for enzyme activity.

  Raju R.V., Datla R.S., Warrington R.C., Sharma R.K.

  Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. Human NMT (hNMT) activity was found to be activated by L-histidin ... >> More

  Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. Human NMT (hNMT) activity was found to be activated by L-histidine in a concentration-dependent manner. In contrast, two structural analogues of L-histidine, L-histidinol and histamine, inhibited hNMT activity in a noncompetitive manner with half-maximal inhibitions of 18 and 1.5 mM, respectively. The inhibition of hNMT activity by L-histidinol was reversed by a 2-fold molar excess of L-histidine, suggesting that L-histidine and L-histidinol were competing for a common site on NMT. Kinetic data indicated that whereas L-histidine enhanced the Vmax, both L-histidinol and histamine decreased the Vmax; none of these compounds altered the Km. Our studies suggest that L-histidine and its analogues may be interacting with His-293, involved in myristoyl-CoA transfer, rather than His-218, and implicated in the transfer of myristoyl-CoA to the peptide substrates. Site-directed mutagenesis of His-293, Val-291, and Glu-290 resulted in proteins with no measurable NMT activity. The most conserved region in the catalytic domain EEVEH (289-293) is critical for the myristoyl-CoA transfer in the NMT-catalyzed reactions. This region will be useful for the design of regulators of NMT function. << Less

  Biochemistry 37:14928-14936(1998) [PubMed] [EuropePMC]

• Kinetic and structural evidence for a sequential ordered Bi Bi mechanism of catalysis by Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase.

  Rudnick D.A., McWherter C.A., Rocque W.J., Lennon P.J., Getman D.P., Gordon J.I.

  The mechanism of catalysis of Escherichia coli-derived Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase (NMT) has been characterized. Previous studies indicated that a high affinity reaction intermediate forms between NMT and myristoyl-CoA in the absence of a peptide substrat ... >> More

  The mechanism of catalysis of Escherichia coli-derived Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase (NMT) has been characterized. Previous studies indicated that a high affinity reaction intermediate forms between NMT and myristoyl-CoA in the absence of a peptide substrate. This complex has been further characterized using S-(2-oxo)pentadecyl-CoA, a nonhydrolyzable myristoyl-CoA analog. Binding studies involving this analog, as well as myristoylpeptide and CoA, have indicated that the CoA moiety of the acyl substrate is retained in the acyl-NMT complex prior to peptide addition. These structural data, along with kinetic studies of myristoylpeptide and CoA product inhibition, indicate that the mechanism of catalysis of NMT is ordered Bi Bi, with myristoyl-CoA binding to NMT occurring prior to peptide binding and CoA release taking place before release of acyl peptide. Further analyses of the interactions between NMT, acyl peptide, and CoA demonstrate that NMT is able to deacylate a myristoylpeptide in the presence of CoA. << Less

  J Biol Chem 266:9732-9739(1991) [PubMed] [EuropePMC]

• N-myristoyltransferase in the leukocytic development processes.

  Kumar S., Singh B., Dimmock J.R., Sharma R.K.

  The lipidic modification of proteins has recently been shown to be of immense importance, although many of the roles of these modifications remain as yet unidentified. One of such key modifications occurring on several proteins is the covalent addition of a 14-carbon long saturated fatty acid, a p ... >> More

  The lipidic modification of proteins has recently been shown to be of immense importance, although many of the roles of these modifications remain as yet unidentified. One of such key modifications occurring on several proteins is the covalent addition of a 14-carbon long saturated fatty acid, a process termed myristoylation. Myristoylation can occur during both co-translational protein synthesis and posttranslationally, confers lipophilicity to protein molecules, and controls protein functions. The protein myristoylation process is catalyzed by the enzyme N-myristoyltransferase (NMT), which exists as two isoforms: NMT1 and NMT2. NMT1 is essential for growth and development, during which rapid cellular proliferation is required, in a variety of organisms. NMT1 is also reported to be elevated in many cancerous states, which also involve rapid cellular growth, albeit in an unwanted and uncontrolled manner. The delineation of myristoylation-dependent cellular functions is still in a state of infancy, and many of the roles of the myristoylated proteins remain to be established. The development of cells of the leukocytic lineage represents a phase of rapid growth and development, and we have observed that NMT1 plays a role in this process. The current review outlines the roles of NMT1 in the growth and differentiation of the cells of leukocytic origin. The described studies clearly demonstrate the roles of NMT1 in the regulation of the developmental processes of the leukocytes cells and provide a basis for further research with the aim of unraveling the roles of protein myristoylation in both cellular and physiological context. << Less

  Cell Tissue Res 345:203-211(2011) [PubMed] [EuropePMC]