The triazine hydrolases : a model system for the evolution of new enzyme function
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2012
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Noor, Sajid
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Abstract
The aim of this study was to use the triazine hydrolases, atrazine chlorohydrolase (AtzA) and melamine deaminase (TriA), as a model system to understand the molecular processes that underpin the emergence of new enzymatic functions. AtzA and TriA differ by just nine amino acid substitutions but have qualitatively different functions. AtzA is a dechlorinase whereas TriA has deaminase as well as some promiscuous dechlorinase activities. A literature review is presented in Chapter 1 that describes existing theories and data on the evolution of new gene functions, with a particular focus on the evolution of microbes that can catabolise otherwise recalcitrant xenobiotics. A review of existing biochemical and genetic data on triazine hydrolases is also included. Chapter 2 describes experiments in which putative evolutionary trajectories between AtzA and TriA were reconstructed and the biochemical and biophysical properties of the intermediate forms were characterised along those trajectories. The order in which the nine amino acid substitutions that separate the enzymes could be introduced productively to either parent enzyme was found to be determined by epistatic interactions between substitutions located within the active site of the enzyme, in particular a serine-cysteine substitution at position 331 and an aspartate-asparagine substitution at position 328. Activity trade-offs were also evident along each trajectory and a strong activity-stability trade-off was observed along trajectories involving increasing melamine deaminase activities. The results of this chapter highlight the role that intramolecular epistasis plays in constraining evolutionary trajectories. Chapter 3 presents the results of an in vitro evolution experiment on an AtzA/TriA intermediate seeking to evolve the enzyme towards greater activities in either direction via random mutagenesis and selection. Various trajectories from the intermediate were analysed, with several mutations not seen in Chapter 2 being recovered which improve either or both of atrazine dechlorinase and melamine deaminase activities without trading off stability. Substitutions at positions 85, 92, 100 and 279 were found to play major roles in some of these new trajectories. Chapter 4 takes a different approach to the issue of AtzA evolution, investigating ongoing evolutionary changes in the system under different selection pressures in natural and laboratory settings since it was first discovered in 1995 (41). This chapter finds that AtzA has continued to evolve in the field, with three further functionally important amino acid changes accumulating among French isolates. One common variant has higher specificity for simazine and greater affinity for a metal ion required for activity. Notably also, ongoing maintenance of the original AtzA - containing Pseudomonad isolate in laboratory culture for 12 years in a medium containing high concentrations of atrazine has led to the fiXation of another amino acid substitution which substantially reduces both activity for s-triazines and thermostability. It is suspected that the high concentrations of atrazine in the medium have relaxed the selection for a highly efficient atrazine dechlorinase activity, but that there is some, as yet uncharacterised, counter-selection for the activity of this enzyme. The general discussion in Chapter 5 includes consideration of several constraints that limit the evolution of new functions. Some future research priorities are also suggested.
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