Structural and mutational characterization of L-carnitine binding to human carnitine acetyltransferase

We report the crystal structure of a binary complex of human peroxisomal carnitine acetyltransferase and the substrate L-carnitine, refined to a resolution of 1.8 Angstrom with an R-factor value of 18.9% (R-free 22.3%). (L)-Carnitine binds to a preformed pocket in the active site tunnel of carnitine acetyltransferase aligned with His(322). The quaternary nitrogen of carnitine forms a pi-cation interaction with Phe(545), while Arg(491) forms an electrostatic interaction with the negatively charged carboxylate group. An extensive hydrogen bond network also occurs between the carboxylate group and Tyr(431), Thr(444), and a bound water molecule. Site-directed mutagenesis and kinetic characterization reveals that Tyr431, Thr(444), Arg(497) and Phe(545) are essential for high affinity binding of L-carnitine. (C) 2004 Elsevier Inc. All rights reserved.