Acetylornithine transcarbamylase: a novel enzyme in arginine biosynthesis.
Ornithine transcarbamylase is a highly conserved enzyme in arginine biosynthesis and the urea cycle. In Xanthomonas campestris, the protein annotated as ornithine transcarbamylase, and encoded by the argF gene, is unable to synthesize citrulline directly from ornithine. We cloned and overexpressed this X. campestris gene in Escherichia coli and show that it catalyzes the formation of N-acetyl-L-citrulline from N-acetyl-L-ornithine and carbamyl phosphate. We now designate this enzyme as an acetylornithine transcarbamylase. The K(m) values for N-acetylornithine and carbamyl phosphate were 1.05 mM and 0.01 mM, respectively. Additional putative transcarbamylases that might also be misannotated were found in the genomes of members of other xanthomonads, Cytophaga, and Bacteroidetes as well as in DNA sequences of bacteria from environmental isolates. It appears that these different paths for arginine biosynthesis arose very early in evolution and that the canonical ornithine transcarbamylase-dependent pathway became the prevalent form. A potent inhibitor, N(alpha)-acetyl-N(delta)-phosphonoacetyl-L-ornithine, was synthesized and showed a midpoint of inhibition at approximately 22 nM; this compound may prove to be a useful starting point for designing inhibitors specific to this novel family of transcarbamylases.
Children's Research Institute, Children's National Medical Center, 111 Michigan Avenue NW, Washington, DC 20010, USA.
Hiroki
H
Morizono
Juan
J
Cabrera-Luque
Dashuang
D
Shi
Rene
R
Gallegos
Saori
S
Yamaguchi
Xiaolin
X
Yu
Norma M
NM
Allewell
Michael H
MH
Malamy
Mendel
M
Tuchman
D001120:Arginine / Q000096:biosynthesis*; D041963:Bacteroidetes / Q000235:genetics; D002221:Carbamyl Phosphate / Q000378:metabolism*; D019878:Carboxyl and Carbamoyl Transferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D003001:Cloning, Molecular; D003589:Cytophaga / Q000235:genetics; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D016680:Genome, Bacterial / Q000235:genetics; D013058:Mass Spectrometry; D015394:Molecular Structure; D009952:Ornithine / Q000031:analogs & derivatives* / Q000378:metabolism; D016959:Xanthomonas campestris / Q000201:enzymology*
D004791:Enzyme Inhibitors; C021951:N(delta)-acetylornithine; D002221:Carbamyl Phosphate; D001120:Arginine; D009952:Ornithine; D019878:Carboxyl and Carbamoyl Transferases; C500792:N-acetylornithine transcarbamylase