The biologically important carnitine biosynthesis pathway in humans proceeds via four enzymatic steps. The first step in carnitine biosynthesis is catalyzed by trimethyllysine hydroxylase (TMLH), a non-heme Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase, which catalyzes the stereospecific hydroxylation of (2S)-N-epsilon-trimethyllysine to (2S, 3S)-3-hydroxy- N-epsilon-trimethyllysine. Here, we report biocatalytic studies on human TMLH and its 19 variants introduced through site-directed mutagenesis. Amino acid substitutions at the sites involved in binding of the Fe(II) cofactor, 2OG cosubstrate and (2S)-N-epsilon-trimethyllysine substrate provide a basic insight into the binding requirements that determine an efficient TMLH-catalyzed conversion of (2S)-Ne-trimethyllysine to (2S, 3S)-3-hydroxy-N-epsilon-trimethyllysine. This work demonstrates the importance of the recognition sites that contribute to the enzymatic activity of TMLH: the Fe(II)-binding H242-D244-H389 residues, R391-R398 involved in 2OG binding and several residues (D231, N334 and the aromatic cage comprised of W221, Y217 and Y234) associated with binding of (2S)-N-epsilon-trimethyllysine.
[1]Radboud Univ Nijmegen, Inst Mol & Mat, Nijmegen, Netherlands.
[2]Jilin Univ, China Japan Union Hosp, Dept Blood Transfus, Jilin, Jilin, Peoples R China.
[3]Radboud Univ Nijmegen, Radboud Inst Mol Life Sci, Med Ctr, Nijmegen, Netherlands.
[4]Univ Southern Denmark, Dept Phys Chem & Pharm, Odense, Denmark.
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