Enzyme promiscuity and the origins of cellular innovations : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Albany, New Zealand

Abstract

Biochemistry textbooks define enzymes as being efficient and highly specific. However, these characteristics are usually associated with a lack of versatility, and therefore, an inability to evolve new functions. In spite of this, it is known that new enzymes can arise rapidly (such as when bacteria evolve antibiotic resistance). One hypothesis proposes that enzymes are actually promiscuous (Jensen, 1976); that is, they are able to carry out secondary reactions, in addition to the one they evolved to catalyze. The goal of this research was to explore the role that promiscuity plays in the origins and evolution of enzyme functions, using Escherichia coli as a model organism. In the first part of this thesis, I report the discovery of two enzymes (alanine racemase and cystathionine ß-lyase) that are reciprocally promiscuous, and are dependent on the cofactor pyridoxal 5’-phosphate (PLP) for activity. In vivo, the cofactor-mediated promiscuous activities of alanine racemase and cystathionine ß-lyase were each successfully improved to near wildtype levels using directed evolution experiments. These results extend Jensen’s hypothesis, and led me to propose that PLP played a significant role in the evolution of new enzymes, in the primordial world. In the second part of the thesis, I developed a comprehensive library-on-library screen to search for E. coli proteins that could mediate improved growth in environments containing either a foreign nutrient or a toxin. Proteins were over-expressed in an attempt to increase their weak, promiscuous activities, and to mimic the common genetic phenomenon of gene amplification. Over-expression of individual proteins conferred improved growth to the host cell in 35% of ~2,000 environments. The findings have important implications for understanding bacterial adaptation to new environments, such as when antibiotic resistance emerges. The ability of promiscuous proteins to drive the emergence of new phenotypes, when their expression is increased, validates the feasibility of the Innovation, Amplification and Divergence (IAD) model for the evolution of new genes (Bergthorsson et al., 2007). Overall, the work described in this thesis demonstrates that protein promiscuity is common, though difficult to predict a priori. My experimental results are consistent with the work of others, in suggesting that promiscuous activities are evolvable. Together, the high frequency and evolvability of promiscuous proteins appear to underpin many different cellular innovations.