Subtilisins

Abstract: Random mutagenesis has been used to engineer the protease subtilisin E to function in a highly nonnatural environment--high concentrations of a polar organic solvent. Sequential rounds of mutagenesis and screening have yielded a variant (PC3) that hydrolyzes a peptide substrate 256 times more efficiently than wild-type subtilisin in 60% dimethylformamide. PC3 subtilisin E and other variants containing different combinations of amino acid substitutions are effective catalysts for transesterification and peptide synthesis in dimethylformamide and other organic media. Starting with a variant containing four effective amino acid substitutions (D60N, D97G, Q103R, and N218S; where, for example, D60N represents Asp-60-->Asn), six additional mutations (G131D, E156G, N181S, S182G, S188P, and T255A) were generated during three sequential rounds of mutagenesis and screening. The 10 substitutions are clustered on one face of the enzyme, near the active site and substrate binding pocket, and all are located in loops that connect core secondary structure elements and exhibit considerable sequence variability in subtilisins from different sources. These variable surface loops are effective handles for "tuning" the activity of subtilisin. Seven of the 10 amino acid substitutions in PC3 are found in other natural subtilisins. Great variability is exhibited among naturally occurring sequences that code for similar three-dimensional structures--it is possible to make use of this sequence flexibility to engineer enzymes to exhibit features not previously developed (or required) for function in vivo.

Abstract: The sequence of the cloned Bacillus amyloliquefaciens subtilisin gene suggested that this secreted serine protease is produced as a larger precursor, designated preprosubtilisin [Wells, J A, Ferrari, E, Henner, D J, Estell, D A & Chen, E Y (1983) Nucleic Acids Res 11, 7911-7925] Biochemical evidence presented here shows that a subtilisin precursor is produced in Bacillus subtilis hosts The precursor is first localized in the cell membrane, reaching a steady-state level of approximately equal to 1000 sites per cell Mutations in the subtilisin gene that alter a catalytically critical residue (ie, aspartate +32----asparagine), or delete the carboxyl-terminal portion of the enzyme that contains catalytically critical residues, block the maturation of this precursor This block occurs when these mutant genes are expressed in B subtilis hosts where the chromosomal subtilisin gene has been deleted When the mutant B amyloliquefaciens subtilisins are expressed in B subtilis hosts that contain an intact chromosomal subtilisin gene, the mutant precursors are processed to a mature form and released to the medium Such processing, in trans, of the precursor is also demonstrated in vitro by addition of active subtilisin Thus, the release of subtilisin from the cell membrane is dependent on an autoproteolytic process that appears to be novel among secreted proteins