Mechanistic insights into the isochorismate pyruvate lyase activity of the catalytically promiscuous PchB from combinatorial mutagenesis and selection.
PchB from Pseudomonas aeruginosa possesses isochorismate pyruvate lyase (IPL) and weak chorismate mutase (CM) activity. Homology modeling based on a structurally characterized CM, coupled with randomization of presumed key active site residues (Arg54, Glu90, Gln91) and in vivo selection for CM activity, was used to derive mechanistic insights into the IPL activity of PchB. Mutation of Arg54 was incompatible with viability, and the CM and IPL activities of an engineered R54K variant were reduced 1,000-fold each. The observation that position 90 was tolerant to substitution but position 91 was essentially confined to Gln or Glu in functional variants rules out involvement of Glu90 in general base catalysis. Counter to the generally accepted mechanistic hypothesis for pyruvate lyases, we propose for PchB a rare [1,5]-sigmatropic reaction mechanism that invokes electrostatic catalysis in analogy to the [3,3]-pericyclic rearrangement of chorismate in CMs. A common catalytic principle for both PchB functions is also supported by the covariance of the catalytic parameters for the CM and IPL activities and the shared functional requirement for a protonated Glu91 in Q91E variants. The experiments demonstrate that focusing directed evolution strategies on the readily accessible surrogate activity of an enzyme can provide valuable insights into the mechanism of the primary reaction.
Künzler
Dominik E
DE
Laboratorium für Organische Chemie, Swiss Federal Institute of Technology (ETH), CH-8093 Zürich, Switzerland.
Sasso
Severin
S
Gamper
Marianne
M
Hilvert
Donald
D
Kast
Peter
P
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D049109:Cell Proliferation; D002826:Chorismate Mutase / Q000737:chemistry; D002942:Circular Dichroism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D019143:Evolution, Molecular; D005786:Gene Expression Regulation; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D005821:Genetic Techniques; D018698:Glutamic Acid / Q000737:chemistry; D005973:Glutamine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D007652:Oxo-Acid-Lyases / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D014764:Viral Proteins / Q000737:chemistry
D001426:Bacterial Proteins; D014764:Viral Proteins; C097122:pchB protein, Pseudomonas aeruginosa; D005973:Glutamine; D018698:Glutamic Acid; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D007652:Oxo-Acid-Lyases; C075960:chorismate pyruvate lyase; D002826:Chorismate Mutase