Carnitine Biosynthesis: Identification of the cDNA Encoding Human -Butyrobetaine Hydroxylase Fre ´de ´ric M. Vaz, Sandy van Gool, Rob Ofman, Lodewijk Ijlst, and Ronald J. A. Wanders Department of Clinical Chemistry and Pediatrics, Academic Medical Center, University of Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands Received August 14, 1998 -Butyrobetaine hydroxylase (EC 1.14.11.1) is the last enzyme in the biosynthetic pathway of L-carnitine and catalyzes the formation of L-carnitine from -butyrobetaine, a reaction dependent on -ketoglu- tarate, Fe 2 , and oxygen. We report the purification of the protein from rat liver to apparent homogeneity, which allowed N-terminal sequencing using Edman degradation. The obtained amino acid sequence was used to screen the expressed sequence tag database and led to the identification of a human cDNA contain- ing an open reading frame of 1161 base pairs encoding a polypeptide of 387 amino acids with a predicted mo- lecular weight of 44.7 kDa. Heterologous expression of the open reading frame in the yeast Saccharomyces cerevisiae confirmed that the cDNA encodes the hu- man -butyrobetaine hydroxylase. Northern blot anal- ysis showed -butyrobetaine hydroxylase expression in kidney (high), liver (moderate), and brain (very low), while no expression could be detected in the other investigated tissues. © 1998 Academic Press Carnitine (3-hydroxy-4-N-trimethylaminobutyrate) is best known for its function in mitochondrial -oxidation since it plays an indispensable role in the transport of activated fatty acids across the mitochon- drial membrane (1, 2). Many organisms, ranging from bacteria to mammals, are able to synthesize carnitine (3–5). In man, carnitine is synthesized in kidney, liver and, presumably, in brain from the essential amino acids lysine and methionine (3, 6). The lysine becomes available as -N-trimethyllysine after degradation of proteins of which lysine residues have been trimethy- lated by a protein-dependent methyl transferase using S-adenosylmethionine as the methyl donor (3). As the first step in carnitine biosynthesis, the -N-trimethyl- lysine is hydroxylated at the 3-position by -N-tri- methyllysine hydroxylase (3, 6). Subsequently, the re- sulting -hydroxy--N-trimethyllysine is cleaved into -trimethylaminobutyraldehyde and glycine by -hydroxy- -N-trimethyllysine aldolase (3, 6), after which the al- dehyde is oxidized by -trimethylaminobutyraldehyde dehydrogenase to yield -butyrobetaine (3, 6, 7). The final step involves the hydroxylation of -butyrobetaine at the 3-position by -butyrobetaine hydroxylase (-BBH) 1 to yield L-carnitine (3, 6, 8). Of all the enzymes of the carnitine biosynthetic path- way, -BBH is the best-studied enzyme. Like -N-tri- methyllysine hydroxylase, -BBH is a non-heme ferrous- iron dioxygenase that requires -ketoglutarate, Fe 2+ and molecular oxygen as cofactors (9). In this class of enzymes, the hydroxylation of the substrate is linked to the oxidative decarboxylation of -ketoglutarate. -BBH has been isolated from various sources includ- ing human kidney (10, 11), calf (12) and rat liver (13, 14) and the bacterium Pseudomonas AK1 (15). The complete amino acid sequence of the Pseudomonas AK1 enzyme has been determined by Edman degrada- tion (16). Although the different enzymatic steps and the in- termediates of the carnitine biosynthesis are well doc- umented, thus far none of the mammalian enzymes has been characterized at the molecular level. We now report the identification of a human cDNA encoding -BBH as demonstrated by heterologous expression in yeast. Our results show that -BBH is expressed in only a few human tissues. MATERIALS AND METHODS Materials. Q-Sepharose, chromatofocusing PBE 94, phenyl Sepharose HP, and 32 P-ATP were obtained from Amersham Phar- macia Biotech (Uppsala, Sweden), -butyrobetaine and carnitine from Sigma (St. Louis, MO), and the Econo-Pac CHT-II hydroxylapa- tite column, from Bio-Rad (Hercules, CA). All other reagents were of 1 Abbreviations used: -BBH, -butyrobetaine hydroxylase; DTT, dithiothreitol; ORF, open reading frame; EST, expressed sequence tag; PCR, polymerase chain reaction; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 250, 506 –510 (1998) ARTICLE NO. RC989343 506 0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.