Site-directed mutagenesis of putative active-site amino acid residues of 3,2-trans-enoyl-CoA isomerase, conserved within the low-homology isomerase/hydratase enzyme family.
During beta-oxidation of unsaturated fatty acids, mitochondrial 3,2-trans-enoyl-CoA isomerase (mECI) converts 3-cis- or 3-trans-enoyl-CoA intermediates into their 2-trans isomers. The cDNA-derived amino acid sequence of mECI shows weak but significant homologies to the peroxisomal trifunctional enzyme (pTFE), the alpha-subunit of the fatty acid degradation complex from Escherichia coli (FadB), the mitochondrial 2-enoyl-CoA hydratase (mECH), the naphthoate synthase encoded by the menB gene from Bacillus subtilis (MenB), and the 4-chlorobenzoyl-CoA dehalogenase from Pseudomonas sp. (CBDH). These proteins from the isomerase/hydratase enzyme family. Tyr-150, Arg-151, and Asp-211 of the mECI are the only amino acids with protic side chains conserved within the enzymes with isomerase activity (pTFE and FadB). These amino acids are exchanged in the remaining enzymes of the isomerase/hydratase family. Glu-165 is conserved in all enzymes with isomerase and/or hydratase activity (pTFE, FadB, and mECH). We argue that these amino acids are possibly involved in the proton transfer at the active site of mECI. To test this hypothesis, mECI was functionally expressed in E. coli. The recombinant enzyme (rmECI) exhibits the same specific activity as the enzyme from rat liver. Exchange of the candidate active-site amino acids by site-directed mutagenesis revealed that Tyr-150 is not involved in isomerase catalysis. The exchange of Arg-151 and Asp-211 leads to a reduced expression of the recombinant enzyme accompanied by a reduced specific activity. The replacement of Glu-165 by Gln leads to a strongly reduced enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Müller-Newen
G
G
Institute of Biochemistry, Medical Faculty, University of Cologne, Germany.
Stoffel
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D019748:Carbon-Carbon Double Bond Isomerases; D017124:Conserved Sequence; D017931:DNA Primers; D064006:Dodecenoyl-CoA Isomerase; D004746:Enoyl-CoA Hydratase / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D007535:Isomerases / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D011994:Recombinant Proteins; D004746:Enoyl-CoA Hydratase; D007535:Isomerases; D019748:Carbon-Carbon Double Bond Isomerases; D064006:Dodecenoyl-CoA Isomerase