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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
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