Emergence of novel enzyme quasi-species depends on the substrate matrix.
Current research on enzyme evolution has shown that many enzymes are promiscuous and have activities with alternative substrates. Mutagenesis tends to relax substrate selectivity, and evolving enzymes can be regarded (summed over evolutionary time) as clusters of enzyme variants, or "quasi-species," tested against a "substrate matrix" defined by all chemical substances to which the evolvants are exposed. In this investigation, the importance of the substrate matrix for identification of evolvable clusters of enzymes was evaluated by random sampling of variants from a library of glutathione transferase (GST) mutants. The variant GSTs were created by DNA shuffling of homologous Alpha class sequences. The substrate matrix was an array of alternative substrates used under defined experimental conditions. The measured enzyme activities produced a rectangular matrix, in which the rows can be projected as enzyme vectors in substrate-activity space and, reciprocally, the columns can be projected as alternative substrate vectors in enzyme-activity space. Multivariate analysis of the catalytic activities demonstrated that the enzyme vectors formed two primary clusters or functional "molecular quasi-species." These quasi-species serve as the raw material from which more specialized enzymes eventually could evolve. The substrate vectors similarly formed two major groups. Identification of separate quasi-species of GSTs in a mutant library was critically dependent on the nature of the substrate matrix. When substrates from just one of the two groups were used, only one cluster of enzymes could be recognized. On the other hand, expansion of the substrate matrix to include additional substrates showed the presence of a third quasi-species among the GST variants already analyzed. Thus, the portrayal of the functional quasi-species is intimately linked to the effective substrate matrix. In natural evolution, the substrates actually encountered therefore play a pivotal role in determining whether latent catalytic abilities become manifest in novel enzymes.
Kurtovic
Sanela
S
Department of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, SE-75123 Uppsala, Sweden.
Shokeer
Abeer
A
Mannervik
Bengt
B
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D016000:Cluster Analysis; D019020:Directed Molecular Evolution; D019143:Evolution, Molecular; D005982:Glutathione Transferase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D019151:Peptide Library; D025341:Principal Component Analysis; D017433:Protein Structure, Secondary; D051381:Rats; D016415:Sequence Alignment; D013379:Substrate Specificity
D050505:Mutant Proteins; D019151:Peptide Library; D005982:Glutathione Transferase