Investigating the active site of human trimethyllysine hydroxylase.

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 (2 S )- N ^ε -trimethyllysine to (2 S ,3 S )-3-hydroxy- N ^ε -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 (2 S )- N ^ε -trimethyllysine substrate provide a basic insight into the binding requirements that determine an efficient TMLH-catalyzed conversion of (2 S )- N ^ε -trimethyllysine to (2 S ,3 S )-3-hydroxy- N ^ε -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 (2 S )- N ^ε -trimethyllysine.
(© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)