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Neutral drift occurring over millions or billions of years results in substantial sequence divergence among enzymes that catalyze the same reaction. Although natural selection maintains the primary activity of orthologous enzymes, there is, by definition, no selective pressure to maintain physiologically irrelevant promiscuous activities. Thus, the levels and the evolvabilities of promiscuous activities may vary among orthologous enzymes. Consistent with this expectation, we have found that the levels of a promiscuous activity in nine gamma-glutamyl phosphate reductase (ProA) orthologs vary by about 50-fold. Remarkably, a single amino acid change from Glu to Ala near the active site appeared to be critical for improvement of the promiscuous activity in every ortholog. The effects of this change varied dramatically. The improvement in the promiscuous activity varied from 50- to 770-fold, and, importantly, was not correlated with the initial level of the promiscuous activity. The decrease in the original activity varied from 190- to 2,100-fold. These results suggest that evolution of a novel enzyme may be possible in some microbes, but not in others. Further, these results underscore the importance of using multiple orthologs as starting points for directed evolution of novel enzyme activities. [ABSTRACT FROM AUTHOR]

Abstract: We devised a growth-based strategy for screening phosphotriesterase mutant libraries for variants with enhanced activity towards organophosphates that generate dimethyl phosphate when hydrolysed. Phosphotriesterase mutants were screened for activity by growing transformed Escherichia coli on agar plates containing methyl paraoxon as a sole phosphorus source. E. coli is capable of growth under these conditions when coexpressing the phosphotriesterase from Agrobacterium radiobacter P230 (OpdA) and the glycerophosphodiester phosphodiesterase from Enterobacter aerogenes (GpdQ). The latter enzyme can hydrolyse the dimethyl phosphate produced by the phosphotriesterase to methyl phosphate, which can then be used by E. coli as a source of phosphate. Phosphotriesterase was expressed from the lac promoter at levels such that its activity was growth-rate limiting. Cultures of the largest colonies (1% of the transformants) were assayed for activity towards paraoxon spectrophotometrically in microtitre plates. This process produced E. coli variants with higher whole cell activity than wild-type, which was found to be a consequence of increased protein expression rather than any increase in enzymatic activity. The mutations present in these mutant enzymes with increased expression were exclusively in the coding region, suggesting the improvement occurs post-transcriptionally. [Copyright &y& Elsevier]

Phosphotriesterases catalyze the hydrolytic detoxification of phosphotriester pesticides and chemical warfare nerve agents with various efficiencies. The directed evolution of phosphotriesterases to enhance the breakdown of poor substrates is desirable for the purposes of bioremediation. A limiting factor in the identification of phosphotriesterase mutants with increased activity is the ability to effectively screen large mutant libraries. To this end, we have investigated the possibility of coupling phosphotriesterase activity to cell growth by using methyl paraoxon as the sole phosphorus source. The catabolism of paraoxon to phosphate would occur via the stepwise enzymatic hydrolysis of paraoxon to dimethyl phosphate, methyl phosphate, and then phosphate. The Escherichia coli strain DH10B expressing the phosphotriesterase from Agrobacterium radiobacter P230 (OpdA) is unable to grow when paraoxon is used as the sole phosphorus source. Enterobacter aerogenes is an organism capable of growing when dimethyl phosphate is the sole phosphorus source. The enzyme responsible for hydrolyzing dimethyl phosphate has been previously characterized as a nonspecific phosphohydrolase. We isolated and characterized the genes encoding the phosphohydrolase operon. The operon was identified from a shotgun clone that enabled E. coli to grow when dimethyl phosphate is the sole phosphorus source. E. coli coexpressing the phosphohydrolase and OpdA grew when paraoxon was the sole phosphorus source. By constructing a short degradative pathway, we have enabled E. coli to use phosphotriesters as a sole source of phosphorus. [ABSTRACT FROM AUTHOR]

Most people believe that new enzymes evolve from existing enzymes because of the conservation of amino acid sequence and tertiary structures in enzymes with different functions and the low probability that such similarities could evolve independently. However, the process by which this divergence occurs is still debatable. A reasonable proposal for this process is the duplication of a gene encoding an existing enzyme, accumulation of mutations in the gene duplicate(s) with consequential functional divergence, followed by selection for an enzyme with a new function . To substantiate this proposal, several areas must be addressed. Which proteins serve as templates? Under what conditions would this series of events occur? What selective advantage does a new enzyme confer upon the host? [Copyright &y& Elsevier]

A method was developed to monitor dynamic changes in protein structure and interfacial behavior on surfaces by single-molecule Förster resonance energy transfer. This method entails the incorporation of unnatural amino acids to site-specifically label proteins with single-molecule Förster resonance energy transfer probes for high-throughput dynamic fluorescence tracking microscopy on surfaces. Structural changes in the enzyme organophosphorus hydrolase (OPH) were monitored upon adsorption to fused silica (FS) surfaces in the presence of BSA on a molecule-by-molecule basis. Analysis of >30,000 individual trajectories enabled the observation of heterogeneities in the kinetics of surface-induced OPH unfolding with unprecedented resolution. In particular, two distinct pathways were observed: a majority population (∼ 85%) unfolded with a characteristic time scale of 0.10 s, and the remainder unfolded more slowly with a time scale of 0.7 s. Importantly, even after unfolding, OPH readily desorbed from FS surfaces, challenging the common notion that surface-induced unfolding leads to irreversible protein binding. This suggests that protein fouling of surfaces is a highly dynamic process because of subtle differences in the adsorption/desorption rates of folded and unfolded species. Moreover, such observations imply that surfaces may act as a source of unfolded (i.e., aggregation-prone) protein back into solution. Continuing study of other proteins and surfaces will examine whether these conclusions are general or specific to OPH in contact with FS. Ultimately, this method, which is widely applicable to virtually any protein, provides the framework to develop surfaces and surface modifications with improved biocompatibility.