Carnitine biosynthesis: identification of the cDNA encoding human gamma-butyrobetaine hydroxylase.
gamma-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 gamma-butyrobetaine, a reaction dependent on alpha-ketoglutarate, Fe2+, 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 containing an open reading frame of 1161 base pairs encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44.7 kDa. Heterologous expression of the open reading frame in the yeast Saccharomyces cerevisiae confirmed that the cDNA encodes the human gamma-butyrobetaine hydroxylase. Northern blot analysis showed gamma-butyrobetaine hydroxylase expression in kidney (high), liver (moderate), and brain (very low), while no expression could be detected in the other investigated tissues.
Vaz
F M
FM
Department of Clinical Chemistry and Pediatrics, Academic Medical Center, University of Amsterdam, The Netherlands.
van Gool
S
S
Ofman
R
R
Ijlst
L
L
Wanders
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002331:Carnitine / Q000096:biosynthesis*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D051381:Rats; D012441:Saccharomyces cerevisiae; D050563:gamma-Butyrobetaine Dioxygenase
D018076:DNA, Complementary; D006899:Mixed Function Oxygenases; D050563:gamma-Butyrobetaine Dioxygenase; D002331:Carnitine