Mammalian myristoyl CoA: protein N-myristoyltransferase

Myristoyl-CoA protein:NMT (N-myristoyl transferase) catalyses the N-myristoylation of cellular proteins with a range of functions and is essential for viability in the protozoan parasites, Leishmania major and Trypanosoma brucei. In our investigations to define the essential downstream targets of NMT, we have focused on the ARF (ADP-ribosylation factor) family of proteins, as growth arrest in Saccharomyces cerevisiae mutants with reduced NMT activity correlates with decreased modification of members of this group of proteins. We have identified nine ARF/ARLs (where ARL stands for ARF-like) encoded in the T. brucei and T. cruzi genomes and ten in L. major. The T. brucei ARL1 protein is expressed only in the mammalian bloodstream form of the parasite, in which it is localized to the Golgi apparatus. RNAi (RNA interference) has been used to demonstrate that ARL1 is essential for viability in these infective cells. Before cell death, depletion of ARL1 protein results in disintegration of the Golgi structure and a delay in exocytosis of the abundant GPI (glycosylphosphatidylinositol)-anchored VSG (variant surface glycoprotein) to the parasite surface.

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Diverse modes of binding in structures ofLeishmania majorN-myristoyltransferase with selective inhibitors

IUCrJ ◽

10.1107/s2052252514013001 ◽

2014 ◽

Vol 1 (4) ◽

pp. 250-260 ◽

Cited By ~ 20

Author(s):

James A. Brannigan ◽

Shirley M. Roberts ◽

Andrew S. Bell ◽

Jennie A. Hutton ◽

Michael R. Hodgkinson ◽

...

Keyword(s):

High Throughput Screening ◽

Active Sites ◽

Screening Program ◽

Peptide Binding ◽

Effective Vaccine ◽

Parasitic Infections ◽

High Morbidity ◽

Peptide Binding Groove ◽

Binding Groove ◽

Myristoyl Coa

The leishmaniases are a spectrum of global diseases of poverty associated with immune dysfunction and are the cause of high morbidity. Despite the long history of these diseases, no effective vaccine is available and the currently used drugs are variously compromised by moderate efficacy, complex side effects and the emergence of resistance. It is therefore widely accepted that new therapies are needed.N-Myristoyltransferase (NMT) has been validated pre-clinically as a target for the treatment of fungal and parasitic infections. In a previously reported high-throughput screening program, a number of hit compounds with activity against NMT fromLeishmania donovanihave been identified. Here, high-resolution crystal structures of representative compounds from four hit series in ternary complexes with myristoyl-CoA and NMT from the closely relatedL. majorare reported. The structures reveal that the inhibitors associate with the peptide-binding groove at a site adjacent to the bound myristoyl-CoA and the catalytic α-carboxylate of Leu421. Each inhibitor makes extensive apolar contacts as well as a small number of polar contacts with the protein. Remarkably, the compounds exploit different features of the peptide-binding groove and collectively occupy a substantial volume of this pocket, suggesting that there is potential for the design of chimaeric inhibitors with significantly enhanced binding. Despite the high conservation of the active sites of the parasite and human NMTs, the inhibitors act selectively over the host enzyme. The role of conformational flexibility in the side chain of Tyr217 in conferring selectivity is discussed.

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Myristoyl-CoA: protein N-myristoyltransferase activity in cancer cells. Purification and characterization of a cytosolic isoform from the murine leukemia cell line L1210

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1993.tb17989.x ◽

1993 ◽

Vol 214 (3) ◽

pp. 853-867 ◽

Cited By ~ 40

Author(s):

Jean A. BOUTIN ◽

Gilles FERRY ◽

Anne-Pascale ERNOULD ◽

Pierrette MAES ◽

Georges REMOND ◽

...

Keyword(s):

Cancer Cells ◽

Cell Line ◽

Leukemia Cell ◽

Leukemia Cell Line ◽

Purification And Characterization ◽

Murine Leukemia Cell ◽

Murine Leukemia ◽

Myristoyl Coa

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Myristic acid auxotrophy caused by mutation of S. cerevisiae myristoyl-CoA:protein N-myristoyltransferase.

The Journal of Cell Biology ◽

10.1083/jcb.113.6.1313 ◽

1991 ◽

Vol 113 (6) ◽

pp. 1313-1330 ◽

Cited By ~ 43

Author(s):

R J Duronio ◽

D A Rudnick ◽

R L Johnson ◽

D R Johnson ◽

J I Gordon

Keyword(s):

Myristic Acid ◽

Fatty Acid Synthetase ◽

Catalytic Efficiency ◽

Kinetic Studies ◽

Fold Increase ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Myristoyl Coa

The S. cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene (NMT1) is essential for vegetative growth. NMT1 was found to be allelic with a previously described, but unmapped and unidentified mutation that causes myristic acid (C14:0) auxotrophy. The mutant (nmt1-181) is temperature sensitive, but growth at the restrictive temperature (36 degrees C) is rescued with exogenous C14:0. Several analogues of myristate with single oxygen or sulfur for methylene group substitutions partially complement the phenotype, while others inhibit growth even at the permissive temperature (24 degrees C). Cerulenin, a fatty acid synthetase inhibitor, also prevents growth of the mutant at 24 degrees C. Complementation of growth at 36 degrees C by exogenous fatty acids is blocked by a mutation affecting the acyl:CoA synthetase gene. The nmt1-181 allele contains a single missense mutation of the 455 residue acyltransferase that results in a Gly451----Asp substitution. Analyses of several intragenic suppressors suggest that Gly451 is critically involved in NMT catalysis. In vitro kinetic studies with purified mutant enzyme revealed a 10-fold increase in the apparent Km for myristoyl-CoA at 36 degrees C, relative to wild-type, that contributes to an observed 200-fold reduction in catalytic efficiency. Together, the data indicate that nmt-181 represents a sensitive reporter of the myristoyl-CoA pools utilized by NMT.

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Characterization of the myristoyl CoA: Glycylpeptide N-myristoyl transferase from rat and bovine brain

Biochemical Society Transactions ◽

10.1042/bst020341s ◽

1992 ◽

Vol 20 (4) ◽

pp. 341S-341S ◽

Cited By ~ 1

Author(s):

R.A. JEFFREY McILHINNEY ◽

KATE McGLONE

Keyword(s):

Bovine Brain ◽

Myristoyl Coa

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Effect of membrane environment on the activity and inhibitability by malonyl-CoA of the carnitine acyltransferase of hepatic microsomal membranes

Biochemical Journal ◽

10.1042/bj3220435 ◽

1997 ◽

Vol 322 (2) ◽

pp. 435-440 ◽

Cited By ~ 12

Author(s):

Neil M. BROADWAY ◽

E. David SAGGERSON

Keyword(s):

Lipid Composition ◽

Membrane Lipids ◽

Specific Activity ◽

Membrane Lipid ◽

Microsomal Membrane ◽

Membrane Lipid Composition ◽

Carnitine Acyltransferase ◽

Malonyl Coa ◽

Microsomal Membranes ◽

Myristoyl Coa

We have investigated the extent to which membrane environment affects the catalytic properties of the malonyl-CoA-sensitive carnitine acyltransferase of liver microsomal membranes. Arrhenius-type plots of activity were linear in the absence and presence of malonyl-CoA (2.5 μM). Sensitivity to malonyl-CoA increased with decreasing assay temperature. Partly purified enzyme displayed an increased K0.5 (substrate concentration supporting half the maximal reaction rate) for myristoyl-CoA and a reduced sensitivity to malonyl-CoA compared with the enzyme in situ in membranes. Reconstitution with liposomes of a range of compositions restored the K0.5 for myristoyl-CoA to values similar to that seen in native membranes. The lipid requirements for restoration of sensitivity to malonyl-CoA were more stringent. When animals were starved for 24 h the specific activity of carnitine acyltransferase in microsomal membrane residues was increased 3.3-fold, whereas sensitivity to malonyl-CoA was decreased to 1/2.8. When enzymes partly purified from fed and starved animals were reconstituted into crude soybean phosphatidylcholine liposomes there was no difference in sensitivity to malonyl-CoA. When partly purified enzyme from fed rats was reconstituted into liposomes prepared from microsomal membrane lipids from fed animals it was 2.2-fold more sensitive to malonyl-CoA than when reconstituted with liposomes prepared from microsomal membrane lipids from starved animals. This suggests that the physiological changes in sensitivity to malonyl-CoA are mediated via changes in membrane lipid composition rather than via modification of the enzyme protein itself. The increased specific actvity of acyltransferase observed on starvation could not be attributed to changes in membrane lipid composition.

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