Engineering New Enzymes

September 02, 1998

The architecture of proteins is intrinsically hierarchic. Secondary structural elements can form motifs or subdomains which assemble to domains. Domains are structurally and functionally independent, where as motifs or subdomains are stabilized by other parts of the protein and are not structurally and functionally independent.

The evolution of proteins has generated numerous functional domains where the active site resides at the boundary of two such subdomains. Consideration of these hierarchical features of the protein architecture by recombination of subdomains from different proteins would therefore open new approaches in the design of novel enzymatic functions and benefit many design problems.

A first attempt in this direction is the recombination of subdomains selected from homologous proteins which have diversified through evolution. In the August 18 issue of Proc. Natl. Acad. Sci. USA (vol. 95, Issue 17, 9813-9818) scientists from the Max Planck Institute of Biochemistry in cooperation with scientists from Boehringer Mannheim GmbH present a method to generate a hybrid gene by combining the gene segment for a subdomain from the digestive proteinase trypsin with a gene segment for a subdomain from the coagulation proteinase factor Xa. The hybrid protein was expressed in E. coli and folded into a functional serine proteinase as shown by X-ray crystallography and enzymatic assays. While its catalytic activity was comparable to that of trypsin and factor Xa, the hybrid protease displayed a much broader substrate specificity than trypsin and factor Xa, a property associated with "unevolved" or "ancient" enzymes.

This strategy allows one now to swap specific subdomain linked functions, to preserve conserved functions at the interface, and to generate new properties at non-conserved or hypervariable surfaces near the interface. Such new surface linked properties—most prominently including binding sites— seem to be ‘unevolved’, and therefore amenable to optimization for specific applications by design or directed evolution.A possible first application of the hybrid is to assist the development of factor Xa directed antitherapeutics. Since factor Xa exhibits a poor ability for crystallization, which considerably delays and hinders determination of co-crystal structures of factor Xa with specific inhibitors, this particular hybrid was chosen as a potential first step in the engineering of a model for fXa-inhibitor interactions with crystallization properties more like trypsin.


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