Gene duplication-divergence is an important driver of metabolic innovation in the plant kingdom. While much is known about sequence/expression evolution of enzyme duplicates over time, how evolution of the actual enzyme activity occurs over time is not clearly understood. In this study, we investigated evolution of functional diversity in enzyme duplicates using the large BAHD acyltransferase family as a model. Comparative genomic analyses and ancestral state reconstruction across multiple sequenced plant genomes revealed significant expansion of the family around the evolution of land plants, rising from 1-3 copies in algal species to dozens to hundreds of copies in angiosperm genomes. To characterize the ancestral state of BAHDs before the expansion, we compiled a database of 140 biochemically characterized members from over 60 plant species as well as assayed activities of 10 BAHDs from multiple species – including charophytic algal species – vs. five different substrate classes known to be utilized by BAHDs. Results were interpreted in the context of a novel substrate similarity network obtained using cheminformatic approaches, and revealed ancestral BAHD promiscuity spread out over a large substrate space comprising of aliphatic and aromatic alcohols and amines. These results also revealed more recent innovations such as anthocyanin/flavonoid, acylsugar, and taxol pathway acyltransferase activities. These findings support the hypothesis that the large BAHD functional diversity is partly a function of ancestral promiscuity, with most duplicates likely eventually specializing in ancestral promiscuous activities. We further investigated the emergence of the novel anthocyanin-acyltransferase activity using MD simulations, site-directed mutagenesis, and enzyme kinetic assays. Our results revealed that a highly conserved tryptophan stabilizes the catalytic histidine and is necessary to maintain the enzyme’s catalytic efficiency. This integrative analysis provides insights into robustness and evolvability of enzyme families, and reveals rules of protein evolution that can help rational engineering of enzymes for synthetic biology.
Coauthors: Jesus Martinez-Gomez – Cornell University;Austin Weigle – University of Illinois at Urbana-Champaign;Gordon Younkin – Boyce Thompson Institute;Jason Chobirko – University of Pittsburg;Alexandra Bennett – Cornell University;Kai Fan – Cornell University;Chelsea Specht – Cornell Univeristy;Diwakar Shukla – University of Illinois at Urbana-Champaign;Gaurav Moghe – Cornell University