Directed cassette mutagenesis

Figure 2.9 Schematic summary of the directed evolution of enantioselective lipase variants originating from the WT PAL used as catalysts in the hydrolytic kinetic resolution of ester rac-1. CMCM = Combinatorial multiple-cassette mutagenesis [8c,22],...

Using X-ray structure data, amino acids which were believed to affect the desired characteristics were identified. Using either site-directed mutagenesis or "cassette" mutagenesis (8) different amino acid substitutions were made in the subtilisin structural gene (2,9). 

Subsequent directed evolution work on Pseudomonas aeruginosa demonstrates that protein sequence space with respect to enantioselectivity is best explored by a three-step procedure (Reetz, 2001) (i) generation of mutants by error-prone PCR at a high-mutation rate (ii) identification of hot regions and spots in the enzyme by error-prone PCR and substantiation by simplified combinatorial multiple-cassette mutagenesis (iii) extension of the process of combinatorial multiple-cassette mutagenesis to cover a defined region of protein sequence space. 

K. -E. Jaeger, Directed evolution of an enantioselective enzyme through combinatorial multiple-cassette mutagenesis, Angew. Chem. Inti Ed. 2001b, 40, 3589-3591. 

A number of other reports concerning in vitro evolution of enzymes have appeared [56, 73], but again application in organic synthesis was seldom the goal. The same applies to studies based on cassette mutagenesis [74]. Nevertheless, these studies are important in other respects, e.g. in the development of methods which may actually be of direct or indirect use to organic chemists. The StEP-method of mutagenesis has been applied successfully in the evolution of an esterase [39]. 

Enzymes are highly specific catalysts. The nature of this specificity is believed to result from structural and electrostatic complementarity between the enzyme and its substrate. The serine protease, subtilisin, is being extensively studied as a model system to explore the effects of single amino acid substitutions on its structure and function Q). The gene for Bacillus amyloliquefaciens subtilisin has been expressed and secreted in B. subtilis 12).A site-directed mutagenesis scheme, cassette mutagenesis ( ), has been used to produce a series of subtilisin variants that are more resistant to oxidants (4), and have altered stability ( ), specificity, and specific activity. 

Outline the procedure of oligonucleotide-directed mutagenesis, including cassette mutagenesis. 

A cassette-replacement approach was used to facilitate the introduction of amino acid mutations at various sites of the thrombin receptor. First, unique endonuclease restriction enzyme sites were generated at several positions within the thrombin receptor cDNA by mutating the nucleotide sequences. Second, the polymerase chain reaction (PCR) with primers encoding for the desired mutations was used to generate the cDNA cassette with the appropriate endonuclease restriction enzyme sites for replacement of the wild-type sequence. The locations for the introduction of the sites were chosen based on two requirements. They needed to be at or near regions of the cDNA sequence that codes for amino acids at junctions of transmembrane domains and extracellular loops. Also, introduction of the sites did not alter the amino acid sequence of the protein. The site-directed mutagenesis method of Kunkel et al.28 was used to introduce the mutations required for generating the... 

He YA, He YQ, Szklarz GD, Halpert JR (1997) Identification of three key residues in substrate recognition site 5 of human cytochrome P450 3A4 by cassette and site-directed mutagenesis. Biochemistry 36, 8831-8839. 

---