Exploring the active site of chorismate mutase by combinatorial mutagenesis and selection: the importance of electrostatic catalysis.
Chorismate mutase (EC 5.4.99.5) catalyzes the intramolecular rearrangement of chorismate to prephenate. Arg-90 in the active site of the enzyme from Bacillus subtilis is in close proximity to the substrate's ether oxygen and may contribute to efficient catalysis by stabilizing the presumed dipolar transition state that would result upon scission of the C--O bond. To test this idea, we have developed a novel complementation system for chorismate mutase activity in Escherichia coli by reengineering parts of the aromatic amino acid biosynthetic pathway. The codon for Arg-90 was randomized, alone and in combination with that for Cys-88, and active clones were selected. The results show that a positively charged residue either at position 88 (Lys) or 90 (Arg or Lys) is essential. Our data provide strong support for the hypothesis that the positive charge is required for stabilization of the transition state of the enzymatic chorismate rearrangement. The new selection system, in conjunction with combinatorial mutagenesis, renders the mechanism of the natural enzyme(s) accessible to further exploration and opens avenues for the improvement of first generation catalytic antibodies with chorismate mutase activity.
Kast
P
P
Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.
Asif-Ullah
M
M
Jiang
N
N
Hilvert
D
D
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002826:Chorismate Mutase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004563:Electrochemistry; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D005816:Genetic Complementation Test; D005818:Genetic Engineering; D014644:Genetic Variation; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D012641:Selection, Genetic
D017931:DNA Primers; D002826:Chorismate Mutase