Converting catabolic ornithine carbamoyltransferase to an anabolic enzyme.
Pseudomonas aeruginosa has an anabolic and a catabolic ornithine carbamoyltransferase (OTCase). In vitro, these homologous enzymes catalyze the same reaction (ornithine + carbamoyl phosphate (CP) in equilibrium citrulline + Pi), yet in vivo they function unidirectionally owing to specific kinetic properties. The catabolic OTC-ase cannot promote the anabolic reaction (citrulline formation) in vivo because of a sigmoidal CP saturation curve and a high CP concentration for half-maximal velocity. The structural basis for this kinetic specialization was examined. The catabolic OTCase lost most of its homotropic cooperativity and gained anabolic activity when an amino acid residue near the CP binding site, Glu-106, was replaced by alanine or glycine. In the anabolic OTCase of Escherichia coli the glutamine residue corresponding to Glu-106 was exchanged for glutamate; however, in this case no CP cooperativity was acquired. Thus, in catabolic OTCase, sequence features in addition to Glu-106 are important for sigmoidal CP saturation, and such a sequence was identified in the C-terminal part. By an in vivo gene fusion technique the 9 C-terminal amino acids of catabolic OTCase were replaced by the homologous 8 amino acids from anabolic OTCase of E. coli; the hybrid enzyme had a markedly reduced homotropic cooperativity. This gene fusion method should be generally useful for directed enzyme evolution.
Baur
H
H
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland.
Tricot
C
C
Stalon
V
V
Haas
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005838:Genotype; D008969:Molecular Sequence Data; D009154:Mutation; D009954:Ornithine Carbamoyltransferase / Q000235:genetics* / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics*; D015183:Restriction Mapping; D014161:Transduction, Genetic
D009954:Ornithine Carbamoyltransferase