Conserved Residues in the Putative Catalytic Triad of Human Bile Acid Coenzyme A:Amino Acid N-Acyltransferase

Sfakianos, Mindan K.; Wilson, Landon; Sakalian, Michael; Falany, Charles N.; Barnes, Stephen

doi:10.1074/jbc.m207463200

Cited by 34 publications

(45 citation statements)

References 31 publications

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“…The catalytic triad of BAAT has been elucidated and involves a cysteine, aspartic acid and histidine, similar to the Type I ACOT enzymes, however the ACOTs contain an active site serine in place of the cysteine in BAAT [131,145]. Interestingly, the ACNATs also contain an active site serine, however this serine is localized in a SerXaaSerXaaGly motif and not in a GlyXaaSerXaaGly motif common to the ACOTs [146].…”

Section: Peroxisomes Contain a Bile-acid Conjugating Enzyme Catalyzinmentioning

confidence: 99%

Novel functions of acyl-CoA thioesterases and acyltransferases as auxiliary enzymes in peroxisomal lipid metabolism

Hunt

Alexson

2008

Progress in Lipid Research

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Section: Peroxisomes Contain a Bile-acid Conjugating Enzyme Catalyzinmentioning

confidence: 99%

Novel functions of acyl-CoA thioesterases and acyltransferases as auxiliary enzymes in peroxisomal lipid metabolism

Hunt

Alexson

2008

Progress in Lipid Research

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“…In short, our studies demonstrate that CPTI is a thiol acyltransferase and that Cys-305 is the essential nucleophilic residue critical for catalysis. In the presence of both substrates, carnitine and palmitoyl-CoA, CPTI acts primarily as an acyltransferase, but in the absence of either carnitine or CoA, CPTI may exhibit low hydrolase activity, resulting in the breakdown of palmitoyl-CoA or palmitoylcarnitine by a mechanism similar to that of the cysteine proteases and hydrolases, which utilize a Cys-His-Asp catalytic triad (33)(34)(35)(36)(37)(38).…”

Section: Cys-305 In M-cpti Is Important For Catalysis 4529mentioning

confidence: 99%

“…His-473 caused a significant loss in CPTI activity (33)(34)(35)(36)(37)(38). As a rate-limiting enzyme that transports long-chain fatty acids from the cytosol to the mitochondrial matrix, CPTI in the presence of carnitine catalyzes the conversion of long-chain acyl-CoAs to acylcarnitines.…”

Section: Cys-305 In M-cpti Is Important For Catalysis 4529mentioning

confidence: 99%

“…We propose a mechanism for the catalysis whereby the active site catalytic base His-473 aided by Asp-454 abstracts a proton from the -SH group of Cys-305 to generate a reactive thiolate anion that carries out a nucleophilic attack on the substrate acyl-CoA, yielding a tetrahedral intermediate that is subsequently converted to a thioacyl-enzyme covalent intermediate (acyl-S-enzyme). It is envisaged that the negative charge from the carboxyl ion of Asp-454 is transferred to His-473 and then to Cys-305 to enhance the power of the nucleophile (33)(34)(35)(36)(37)(38). Deacylation and transfer of the acyl group to carnitine occur by a nucleophilic attack of the thioester bond of the acyl-S-enzyme intermediate with the reactive carnitine oxyanion generated by abstraction of a proton from the hydroxyl group of carnitine by the general base catalyst at the active site, His-473, assisted by Asp-454, resulting in the formation of a second tetrahedral intermediate that breaks down to yield the product acylcarnitine and regenerate the enzyme as shown in Fig.…”

Section: Cys-305 In M-cpti Is Important For Catalysis 4529mentioning

confidence: 99%

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Cysteine-scanning Mutagenesis of Muscle Carnitine Palmitoyltransferase I Reveals a Single Cysteine Residue (Cys-305) Is Important for Catalysis

Liu¹,

Zheng²,

Treber

et al. 2005

Journal of Biological Chemistry

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Carnitine palmitoyltransferase (CPT) I catalyzes the conversion of long-chain fatty acyl-CoAs to acyl carnitines in the presence of L-carnitine, a rate-limiting step in the transport of long-chain fatty acids from the cytoplasm to the mitochondrial matrix. To determine the role of the 15 cysteine residues in the heart/skeletal muscle isoform of CPTI (M-CPTI) on catalytic activity and malonyl-CoA sensitivity, we constructed a 6-residue N-terminal, a 9-residue C-terminal, and a 15-residue cysteineless M-CPTI by cysteine-scanning mutagenesis. Both the 9-residue C-terminal mutant enzyme and the complete 15-residue cysteineless mutant enzyme are inactive but that the 6-residue N-terminal cysteineless mutant enzyme had activity and malonyl-CoA sensitivity similar to those of wild-type M-CPTI. Mutation of each of the 9 C-terminal cysteines to alanine or serine identified a single residue, Cys-305, to be important for catalysis. Substitution of Cys-305 with Ala in the wild-type enzyme inactivated M-CPTI, and a single change of Ala-305 to Cys in the 9-residue C-terminal cysteineless mutant resulted in an 8-residue C-terminal cysteineless mutant enzyme that had activity and malonyl-CoA sensitivity similar to those of the wild type, suggesting that Cys-305 is the residue involved in catalysis. Sequence alignments of CPTI with the acyltransferase family of enzymes in the GenBank TM led to the identification of a putative catalytic triad in CPTI consisting of residues Cys-305, Asp-454, and His-473. Based on the mutagenesis and substrate labeling studies, we propose a mechanism for the acyltransferase activity of CPTI that uses a catalytic triad composed of Cys-305, His-473, and Asp-454 with Cys-305 serving as a probable nucleophile, thus acting as a site for covalent attachment of the acyl molecule and formation of a stable acyl-enzyme intermediate. This would in turn allow carnitine to act as a second nucleophile and complete the acyl transfer reaction.Carnitine palmitoyltransferase (CPT) 1 I catalyzes the conversion of long-chain fatty acyl-CoAs to acyl carnitines in the presence of L-carnitine, a rate-limiting step in the transport of long-chain fatty acids from the cytoplasm to the mitochondrial matrix (1, 2). Mammalian tissues express two isoforms of CPTI, a liver isoform (L-CPTI) and a heart/skeletal muscle isoform (M-CPTI), which are 62% identical in amino acid sequence (3-8). As an enzyme that catalyzes the first rate-limiting step in fatty acid oxidation, CPTI is regulated by its physiological inhibitor, malonyl-CoA, the first intermediate in fatty acid synthesis, suggesting a coordinated control of fatty acid synthesis and oxidation (1, 2). Previous studies by our laboratory and others have established that M-CPTI is more sensitive to malonyl-CoA inhibition than L-CPTI (3-8). The molecular/ structural basis for the differences in malonyl-CoA sensitivity between M-CPTI and L-CPTI was established recently by our demonstration that substitution of the conserved Cterminal L-CPTI residue Glu-590 with alanine increased...

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“…BAT is responsible for the conjugation of bile acids with amino acids. This is a two-step reaction in which bile acids are first converted to CoA thioesters by bile acid CoA ligase and are then conjugated with an amino acid by BAT (23,24).…”

mentioning

confidence: 99%

Inactivation of human liver bile acid CoA:amino acid N-acyltransferase by the electrophilic lipid, 4-hydroxynonenal

Shonsey

Eliuk

Johnson

et al. 2008

Journal of Lipid Research

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The hepatic enzyme bile acid CoA:amino acid N-acyltransferase (BAT) catalyzes the formation of amino acid-conjugated bile acids. In the present study, protein carbonylation of BAT, consistent with modification by reactive oxygen species and their products, was increased in hepatic homogenates of apolipoprotein E knock-out mice. 4-Hydroxynonenal (4HNE), an electrophilic lipid generated by oxidation of polyunsaturated long-chain fatty acids, typically reacts with the amino acids Cys, His, Lys, and Arg to form adducts, some of which (Michael adducts) preserve the aldehyde (i.e., carbonyl) moiety. Because two of these amino acids (Cys and His) are members of the catalytic triad of human BAT, it was proposed that 4HNE would cause inactivation of this enzyme. As expected, human BAT (1.6 mM) was inactivated by 4HNE in a dose-dependent manner. To establish the sites of 4HNE's reaction with BAT, peptides from proteolysis of 4HNE-treated, recombinant human BAT were analyzed by peptide mass fingerprinting and by electrospray ionizationtandem mass spectrometry using a hybrid linear ion trap Fourier transform-ion cyclotron resonance mass spectrometer. The data revealed that the active-site His (His362) dosedependently formed a 4HNE adduct, contributing to loss of activity, although 4HNE adducts on other residues may also contribute.

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Conserved Residues in the Putative Catalytic Triad of Human Bile Acid Coenzyme A:Amino Acid N-Acyltransferase

Cited by 34 publications

References 31 publications

Novel functions of acyl-CoA thioesterases and acyltransferases as auxiliary enzymes in peroxisomal lipid metabolism

Novel functions of acyl-CoA thioesterases and acyltransferases as auxiliary enzymes in peroxisomal lipid metabolism

Cysteine-scanning Mutagenesis of Muscle Carnitine Palmitoyltransferase I Reveals a Single Cysteine Residue (Cys-305) Is Important for Catalysis

Inactivation of human liver bile acid CoA:amino acid N-acyltransferase by the electrophilic lipid, 4-hydroxynonenal

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