RESUMO
Mycobacteria contain a large number of highly divergent species and exhibit unusual lipid metabolism profiles, believed to play important roles in immune invasion. Thioesterases modulate lipid metabolism through the hydrolysis of activated fatty-acyl CoAs; multiple copies are present in mycobacteria, yet many remain uncharacterized. Here, we undertake a comprehensive structural and functional analysis of a TesB thioesterase from Mycobacterium avium (MaTesB). Structural superposition with other TesB thioesterases reveals that the Asp active site residue, highly conserved across a wide range of TesB thioesterases, is mutated to Ala. Consistent with these structural data, the wild-type enzyme failed to hydrolyze an extensive range of acyl-CoA substrates. Mutation of this residue to an active Asp residue restored activity against a range of medium-chain length fatty-acyl CoA substrates. Interestingly, this Ala mutation is highly conserved across a wide range of Mycobacterium species but not found in any other bacteria or organism. Our structural homology analysis revealed that at least one other TesB acyl-CoA thioesterase also contains an Ala residue at the active site, while two other Mycobacterium TesB thioesterases harbor an Asp residue at the active site. The inactive TesBs display a common quaternary structure that is distinct from that of the active TesB thioesterases. Investigation of the effect of expression of either the catalytically active or inactive MaTesB in Mycobacterium smegmatis exposed, to the best of our knowledge, the first genotype-phenotype association implicating a mycobacterial tesB gene. This is the first report that mycobacteria encode active and inactive forms of thioesterases, the latter of which appear to be unique to mycobacteria.
Assuntos
Acil Coenzima A/química , Proteínas de Bactérias/química , Mycobacterium avium/enzimologia , Mycobacterium smegmatis/enzimologia , Palmitoil-CoA Hidrolase/química , Acil Coenzima A/metabolismo , Alanina/química , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Estudos de Associação Genética , Hidrólise , Isoenzimas/química , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Mutação , Mycobacterium avium/genética , Mycobacterium smegmatis/genética , Palmitoil-CoA Hidrolase/classificação , Palmitoil-CoA Hidrolase/genética , Palmitoil-CoA Hidrolase/metabolismo , Domínios Proteicos , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/classificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
We have previously reported that long chain acyl-CoA thioesterase activity was induced about 10-fold in rat liver mitochondria, when treating rats with the peroxisome proliferator di(2-ethylhexyl)phthalate (Wilcke M., and Alexson S. E. H (1994) Eur. J. Biochem. 222, 803-811). Here we have characterized two enzymes which are responsible for the majority of long chain acyl-CoA thioesterase activity in mitochondria from animals treated with peroxisome proliferators. A 40-kDa enzyme was purified and characterized as a very long chain acyl-CoA thioesterase (MTE-I). The second enzyme was partially purified and characterized as a long chain acyl-CoA thioesterase (MTE-II). MTE-I was inhibited by p-chloromercuribenzoic acid, which implicates the importance of a cysteine residue in, or close, to the active site. Antibodies against MTE-I demonstrated the presence of immunologically related acyl-CoA thioesterases in peroxisomes and cytosol. High expression of MTE-I was found in liver from peroxisome proliferator treated rats and in heart and brown fat from control and induced rats. Comparison of physical and catalytical characteristics of the enzymes studied here and previously purified acyl-CoA thioesterases suggest that MTE-I and MTE-II are novel enzymes.