A novel L-amino acid ligase from Bacillus subtilis NBRC3134, a microorganism producing peptide-antibiotic rhizocticin.
L-amino acid ligase catalyzes the formation of an alpha-peptide bond from unprotected L-amino acids in an ATP-dependent manner, and this enzyme is very useful in efficient peptide production. We performed enzyme purification to obtain a novel L-amino acid ligase from Bacillus subtilis NBRC3134, a microorganism producing peptide-antibiotic rhizocticin. Rhizocticins are dipeptide or tripeptide antibiotics and commonly possess L-arginyl-L-2-amino-5-phosphono-3-cis-pentenoic acid. The purification was carried out by detecting L-arginine hydroxamate synthesis activity, and a target enzyme was finally purified 1,280-fold with 0.8% yield. The corresponding gene was then cloned and designated rizA. rizA was 1,242 bp and coded for 413 amino acid residues. Recombinant RizA was prepared, and it was found that the recombinant RizA synthesized dipeptides whose N-terminus was L-arginine in an ATP-dependent manner. RizA had strict substrate specificity toward L-arginine as the N-terminal substrate; on the other hand, the substrate specificity at the C-terminus was relaxed.
Kino
Kuniki
K
Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan. kkino@waseda.jp
Kotanaka
Yoichi
Y
Arai
Toshinobu
T
Yagasaki
Makoto
M
D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000378:metabolism; D003001:Cloning, Molecular; D004151:Dipeptides / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D008025:Ligases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000096:biosynthesis*; D009943:Organophosphorus Compounds; D010748:Phosphopeptides / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D000900:Anti-Bacterial Agents; D004151:Dipeptides; D009842:Oligopeptides; D009943:Organophosphorus Compounds; D010748:Phosphopeptides; D011994:Recombinant Proteins; C541525:isoleucyl-arginyl-2-amino-5-phosphono-3-pentenoic acid; C541526:leucyl-arginyl-2-amino-5-phosphono-3-pentenoic acid; C096640:rhizocticin B; C063847:rhizocticin A; D008025:Ligases