The Evolutionary Accessibility of New Enzymes Functions: A Case Study from the Biotin Pathway

Enzymes group naturally into families according to similarity of sequence, structure, and underlying mechanism. Enzymes belonging to the same family are considered to be homologs --the products of evolutionary divergence, whereby the first family member provided a starting point for conversions to new but related functions. In fact, despite their similarities, these families can include remarkable functional diversity. Here we focus not on minor functional variations within families, but rather on innovations --transitions to genuinely new catalytic functions. Prior experimental attempts to reproduce such transitions have typically found that many mutational changes are needed to achieve even weak functional conversion, which raises the question of their evolutionary feasibility. To further investigate this, we examined the members of a large enzyme superfamily, the PLP-dependent transferases, to find a pair with distinct reaction chemistries and high structural similarity. We then set out to convert one of these enzymes, 2-amino-3-ketobutyrate CoA ligase (Kbl 2 ), to perform the metabolic function of the other, 8-amino-7-oxononanoate synthase (BioF 2 ). After identifying and testing 29 amino acid changes, we found three groups of active-site positions and one single position where Kbl 2 side chains are incompatible with BioF 2 function. Converting these side chains in Kbl 2 makes the residues in the active-site cavity identical to those of BioF 2 , but nonetheless fails to produce detectable BioF 2 -like function in vivo . We infer from the mutants examined that successful functional conversion would in this case require seven or more nucleotide substitutions. But evolutionary innovations requiring that many changes would be extraordinarily rare, becoming probable only on timescales much longer than the age of life on earth. Considering that Kbl 2 and BioF 2 are judged to be close homologs by the usual similarity measures, this result and others like it challenge the conventional practice of inferring from similarity alone that transitions to new functions occurred by Darwinian evolution. [See published correction to this paper:  doi:10.5048/BIO-C.2011.1.e1 ].