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Characterization of novel microbial transcarbamylases
Author(s) -
CabreraLuque Juan,
Tuchman Mendel,
Morizono Hiroki
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1013-d
Subject(s) - ornithine transcarbamylase , ornithine , biosynthesis , biochemistry , enzyme , ornithine carbamoyltransferase , arginine , carbamyl phosphate , biology , chemistry , urea cycle , amino acid
Ornithine transcarbamylase (OTCase) is an essential enzyme for the de novo biosynthesis of arginine. Several microbial genome sequences were found to contain OTCase‐like genes lacking a highly conserved Ser‐Met‐Gly motif. These genes from Xanthomonas campestris and Bacteroides fragilis were cloned and expressed in E. coli and purified. Enzymatic activity assays, nuclear magnetic resonance (NMR) and liquid chromatography‐mass spectroscopy (LC‐MS) showed that these enzymes utilized N‐Acetyl‐L‐Ornithine (AO) and N‐Succinyl‐L‐Ornithine (SO), respectively. N‐Acetyl‐L‐Ornithine transcarbamylase (AOTCase) had a Km for AO of 0.14mM, while that of N‐Succinyl‐L‐Ornithine transcarbamylase (SOTCase) for SO was 0.238mM. In addition, SOTCase shows pronounced substrate inhibition while AOTCase does not. Two compounds, N‐Acetyl or N‐Succinyl‐phosphoacetyl‐L‐Ornithine (PALAO or PALSO) analogous to a bisubstrate inhibitor of OTCase were synthesized and tested. PALAO inhibited AOTCase but not OTCase or SOTCase. PALSO does not have any effect on AOTCase or OTCase while inhibiting SOTCase. In these organisms, deacylation must follow transcarbamylation; a reversal of the steps in the canonical arginine biosynthetic pathway. In addition, for B. fragilis , the high specificity for SO implies that arginine biosynthesis proceeds via succinylated intermediates instead of acetylated ones.