Probing the role of the C-terminus of Bacillus subtilis chorismate mutase by a novel random protein-termination strategy.
A novel strategy combining random protein truncation and genetic selection has been developed to identify dispensable C-terminal segments of an enzyme. This approach, which entails the random introduction of premature termination codons, was applied to the last 17 residues of chorismate mutase from Bacillus subtilis (BsCM). Although structurally ill-defined, the C-terminus of BsCM has been proposed to cap the active site upon substrate binding and affect catalysis. However, sequence patterns of 178 selected gene variants show that the final 11 residues of the protein can be mutated and even removed without significantly impairing activity in vivo. In fact, none of the randomized residues is absolutely required, but a preference for wild-type Lys111, Ala112, Leu115, and Arg116 is apparent. These residues are part of a C-terminal 3(10)-helix and provide contacts with the rest of the protein or its ligands. The kinetic parameters of selected enzyme variants show that truncations and mutations do not significantly impair catalytic turnover (k(cat)) but substantially decrease k(cat)/K(m). Thus, while the 17 C-terminal residues of BsCM do not participate directly in the chemical rearrangement, they appear to contribute to enzymatic efficiency via uniform binding of the substrate and transition state.
Gamper
M
M
Laboratorium für Organische Chemie, Swiss Federal Institute of Technology (ETH), ETH-Zentrum, Universitätstrasse 16, CH-8092 Zürich, Switzerland.
Hilvert
D
D
Kast
P
P
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D002384:Catalysis; D002826:Chorismate Mutase / Q000138:chemical synthesis / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D015723:Gene Library; D018390:Gene Targeting; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D017384:Sequence Deletion
D010446:Peptide Fragments; D002826:Chorismate Mutase