Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities.
The isochorismate and salicylate synthases are members of the MST family of enzymes. The isochorismate synthases establish an equilibrium for the conversion chorismate to isochorismate and the reverse reaction. The salicylate synthases convert chorismate to salicylate with an isochorismate intermediate; therefore, the salicylate synthases perform isochorismate synthase and isochorismate-pyruvate lyase activities sequentially. While the active site residues are highly conserved, there are two sites that show trends for lyase-activity and lyase-deficiency. Using steady state kinetics and HPLC progress curves, we tested the "interchange" hypothesis that interconversion of the amino acids at these sites would promote lyase activity in the isochorismate synthases and remove lyase activity from the salicylate synthases. An alternative, "permute" hypothesis, that chorismate-utilizing enzymes are designed to permute the substrate into a variety of products and tampering with the active site may lead to identification of adventitious activities, is tested by more sensitive NMR time course experiments. The latter hypothesis held true. The variant enzymes predominantly catalyzed chorismate mutase-prephenate dehydratase activities, sequentially generating prephenate and phenylpyruvate, augmenting previously debated (mutase) or undocumented (dehydratase) adventitious activities.
Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, United States.
Kathleen M
KM
Meneely
Qianyi
Q
Luo
Audrey L
AL
Lamb
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D006860:Hydrogen Bonding; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008190:Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D012459:Salicylates / Q000378:metabolism; D017262:Siderophores / Q000096:biosynthesis*; D014364:Tryptophan / Q000096:biosynthesis*; D024482:Vitamin K 2 / Q000378:metabolism*
D001426:Bacterial Proteins; D012459:Salicylates; D017262:Siderophores; D024482:Vitamin K 2; D014364:Tryptophan; D008190:Lyases; C022620:salicylate synthetase; D019751:Intramolecular Transferases; C097120:PchA protein, Pseudomonas aeruginosa
Enzyme engineering; Isochorismate synthase; Salicylate synthase; Siderophore biosynthesis