In essence, evolutionary processes occur gradually, while maintaining fitness throughout. Along this line, it has been proposed that the ability of a progenitor to promiscuously catalyze a low level of the evolving activity could facilitate the divergence of a new function by providing an immediate selective advantage. To directly establish a role for promiscuity in the divergence of natural enzymes, we attempted to trace the origins of a bacterial phosphotriesterase (PTE), an enzyme thought to have evolved for the purpose of degradation of a synthetic insecticide introduced in the 20th century. We surmised that PTE's promiscuous lactonase activity may be a vestige of its progenitor and tested homologues annotated as "putative PTEs" for lactonase and phosphotriesterase activity. We identified three genes that define a new group of microbial lactonases dubbed PTE-like lactonases (PLLs). These enzymes proficiently hydrolyze various lactones, and in particular quorum-sensing N-acyl homoserine lactones (AHLs), and exhibit much lower promiscuous phosphotriesterase activities. PLLs share key sequence and active site features with PTE and differ primarily by an insertion in one surface loop. Given their biochemical and biological function, PLLs are likely to have existed for many millions of years. PTE could have therefore evolved from a member of the PLL family while utilizing its latent promiscuous paraoxonase activity as an essential starting point.

To efficiently catalyze a chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex, the chemical reaction and product release. These distinct demands could be satisfied via fluctuation between different conformational substates (CSs) with unique configurations and catalytic properties. However, there is debate as to how these rapid conformational changes, or dynamics, exactly affect catalysis. As a model system, we have studied bacterial phosphotriesterase (PTE), which catalyzes the hydrolysis of the pesticide paraoxon at rates limited by a physical barrier-either substrate diffusion or conformational change. The mechanism of paraoxon hydrolysis is understood in detail and is based on a single, dominant, enzyme conformation. However, the other aspects of substrate turnover (substrate binding and product release), although possibly rate-limiting, have received relatively little attention. This work identifies ''open'' and ''closed'' CSs in PTE and dominant structural transition in the enzyme that links them. The closed state is optimally preorganized for paraoxon hydrolysis, but seems to block access to/from the active site. In contrast, the open CS enables access to the active site but is poorly organized for hydrolysis. Analysis of the structural and kinetic effects of mutations distant from the active site suggests that remote mutations affect the turnover rate by altering the conformational landscape.dynamics ͉ enzyme catalysis ͉ evolution ͉ conformational fluctuation A catalyst is defined as a molecule that increases the rate of a chemical reaction by providing an alternative pathway of lower activation energy. A second, sometimes overlooked, requirement of a catalyst is that it is not consumed during the reaction, i.e., it must turn over multiple reactions. With reported rate enhancements of up to 10 17 (1) and turnover rates reaching 10 4 s Ϫ1 (2), enzymes are truly extraordinary catalysts. In addition to efficient rate enhancement of the chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex and product release. So, how does a single protein sequence satisfy these different demands?Our understanding of the mechanisms by which the active sites of enzymes lower the activation energy for various chemical reactions has developed steadily in the past decades and it is clear that the specific organization of chemical groups within the active sites of enzymes provides an electrostatic environment that is markedly different to the conditions in which the uncatalyzed reaction occurs, and results in remarkable rate enhancements (3). There have also been a number of studies showing that transitions between conformational substates (CSs) (4, 5) on a variable energy landscape (6, 7) are an integral part of the full catalytic cycles, or substrate turnover, in many enzymes (7-12). Despite much progress, there is some debate surrounding the exact role that such transitions, or dynamics, play in enzymatic catalysis (7, 13). The deb...

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