Identification of emerging quasi-species in directed enzyme evolution

The bases of enzyme evolution are structural changes in protein scaffolds combined with recognition and propagation of novel variants with valuable functional properties. Structural diversification may be accomplished by a variety of methods, including random mutations, homologous recombinations, and insertions and deletions of coding DNA sequences. The functional consequences of mutations are manifested at the protein level and are dependent on a substrate matrix, when catalytic properties are requested. Libraries of variant enzymes showing promiscuous activities can be interrogated with a set of alternative substrates. We demonstrate using a library of glutathione transferases (GSTs) that the functional properties are not uniformly distributed in substrate-activity space but form clusters, or quasi-species. Multivariate analysis facilitates the identification of such quasi-species, which can be regarded as the proper developing units in molecular evolution.