Environmental selection and epistasis in an empirical phenotype-environment-fitness landscape.

Fitness landscapes, mappings of genotype/phenotype to their effects on fitness, are invaluable concepts in evolutionary biochemistry. Although widely discussed, measurements of phenotype-fitness landscapes in proteins remain scarce. Here, we quantify all single mutational effects on fitness and phenotype (EC50) of VIM-2 ß-lactamase across a 64-fold range of ampicillin concentrations. We then construct a phenotype-fitness landscape that takes variations in environmental selection pressure into account. We found that a simple, empirical landscape accurately models the ~39,000 mutational data points, suggesting that the evolution of VIM-2 can be predicted on the basis of the selection environment. Our landscape provides new quantitative knowledge on the evolution of the ß-lactamases and proteins in general, particularly their evolutionary dynamics under subinhibitory antibiotic concentrations, as well as the mechanisms and environmental dependence of non-specific epistasis.

Evolution of Enzyme Superfamilies: Comprehensive Exploration of Sequence-Function Relationships.

The sequence and functional diversity of enzyme superfamilies have expanded through billions of years of evolution from a common ancestor. Understanding how protein sequence and functional "space" have expanded, at both the evolutionary and molecular level, is central to biochemistry, molecular biology, and evolutionary biology. Integrative approaches that examine protein sequence, structure, and function have begun to provide comprehensive views of the functional diversity and evolutionary relationships within enzyme superfamilies. In this review, we outline the recent advances in our understanding of enzyme evolution and superfamily functional diversity. We describe the tools that have been used to comprehensively analyze sequence relationships and to characterize sequence and function relationships. We also highlight recent large-scale experimental approaches that systematically determine the activity profiles across enzyme superfamilies. We identify several intriguing insights from this recent body of work. First, promiscuous activities are prevalent among extant enzymes. Second, many divergent proteins retain "function connectivity" via enzyme promiscuity, which can be used to probe the evolutionary potential and history of enzyme superfamilies. Finally, we discuss open questions regarding the intricacies of enzyme divergence, as well as potential research directions that will deepen our understanding of enzyme superfamily evolution.

Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase.

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.

Effects of amino acid substitution on the physicochemical properties of artificial proteins with random sequences.

Physicochemical properties of four random proteins, each consisting of about 150 amino acid residues with different sequence identity, were compared to know the correlation between the physicochemical properties and its sequence. The results showed that the extent of the sequence alterations correlated well with the extent of differences in CD spectra, roughly with those in pH-solubility profiles and sedimentation velocity, and not with that in the binding of a hydrophobic fluorescent dye (ANS). Therefore, proteins with similar sequences can have different physicochemical properties, indicating that the extent of mutational effects varies in response to the sequence being altered. This warrants the evolution of a protein in a sequence-specific manner.