کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1948801 1537493 2006 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Mutation of Lys242 allows Δ3-Δ2-enoyl-CoA isomerase to acquire enoyl-CoA hydratase activity
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Mutation of Lys242 allows Δ3-Δ2-enoyl-CoA isomerase to acquire enoyl-CoA hydratase activity
چکیده انگلیسی

We report here a novel example of generating hydratase activity through site-directed mutagenesis of a single residue Lys242 of rat liver mitochondrial Δ3-Δ2-enoyl-CoA isomerase, which is one of the key enzymes involved in fatty acid oxidation and a member of the crotonase superfamily. Lys242 is at the C-terminal of the enzyme, which is far from the active site in the crotonase superfamily and forms a salt bridge with Asp149. A variety of mutant expression plasmids were constructed, and it was observed that mutation of Lys242 to nonbasic residues allowed the mutants to have enoyl-CoA hydratase activity confirmed by HPLC analysis of the incubation mixture. Kinetic studies of these mutants were carried out for both isomerase and hydratase activities. Mutant K242C showed a kcat value of 1.0 s− 1 for hydration reaction. This activity constitutes about 10% of the total enzyme activity, and the remaining 90% is its natural isomerase activity. To the best of our knowledge, this is the first report on the generation of functional promiscuity through single amino acid mutation far from the active site. This may be a simple and efficient approach to designing a new enzyme based on an existing template. It could perhaps become a general methodology for facilitating an enzyme to acquire a type enzymatic activity that belongs to another member of the same superfamily, by interrupting a key structural element in order to introduce ambiguity, using site-directed mutagenesis.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochimica et Biophysica Acta (BBA) - General Subjects - Volume 1760, Issue 12, December 2006, Pages 1874–1883
نویسندگان
, , , , , ,