Emergence of diverse biochemical activities in evolutionarily conserved structural scaffolds of proteins

Abstract

Comparative analysis of numerous protein structures that have become available in the past few years, combined with genome comparison, has yielded new insights into the evolution of enzymes and their functions. In addition to the well-known diversification of substrate specificities, enzymes with several widespread catalytic folds, particularly the TIM barrel, the RRM-like domain and the double-stranded β-helix (cupin) domain, have been extensively explored in ‘reaction space’, resulting in the evolution of numerous, diverse catalytic activities supported by the same structural scaffold. Common protein folds differ widely in the diversity of catalyzed reactions. The biochemical plasticity of a fold seems to hinge on the presence of a generic, symmetrical substrate-binding pocket as opposed to highly specialized binding sites.

Introduction

On the basis of the sequences and structures of a limited set of proteins, Zuckerkandl and Pauling [1] proposed, almost 40 years ago, that proteins with disparate biochemical properties could evolve from a single ancestral protein. The accumulation of protein sequence and structure data since their pioneering work has only reinforced their contention, making it one of the central tenets of evolutionary biology. Subsequent analysis of sequences and structures has shown that the biochemical mechanism of catalysis typically relies on a constellation of a few amino acid residues that are embedded in a distinct globular domain. The rest of the globular domain functions as a structural scaffold supporting the catalytic centre and also contributes to interactions with the substrates, co-factors and other proteins. This principle had been extensively exploited by natural selection to generate the diversity seen in the universe of enzymes from a relatively small number of ancestral enzymes. Generally, the set of catalytic residues is highly conserved during evolution, whereas variations tend to occur in the substrate-binding and cofactor-binding sites. This results in families of enzymes exploring ‘substrate space’ by adapting essentially the same biochemical activity on a range of different substrates. Another common variation on this theme is the fusion of various accessory domains to the catalytic domain, which results in diversity of allosteric regulation [2^•] or cellular localization of the enzyme without changing the catalytic activity.

Analysis of the numerous, recently amassed protein crystal structures revealed other, less-obvious principles that govern evolutionary diversification of enzymes. On several occasions, enzymes with similar catalytic residues have been shown to explore considerable diversity in ‘reaction space’. For example, sequence and structure comparisons indicated that bacterial DnaG-type primases and most topoisomerases (except the topoisomerase IB) share an evolutionarily related catalytic domain (the so-called TOPRIM domain) with an identical core set of catalytic residues 3., 4., 5.. Thus, two distinct types of reactions, namely nucleotidyltransferase (transferase; class 2 according to the EC classification) and topoisomerase (isomerase; class 5 according to the EC classification), have evolved within the same family of enzymes. However, because these reactions involve manipulations of the same phosphoester bonds in similar substrates (nucleotides or polynucleotides), it is easy to conceive their common origin and dependence on the same set of catalytic residues. A more dramatic illustration of the exploration of the reaction space emerged from the crystal structures of several enzymes in the stem glycolytic (Embden–Meyerhoff) pathway: four of the glycolytic enzymes, 1,6 fructose bisphosphate aldolase, triose phosphate isomerase, enolase and pyruvate kinase, that catalyse three distinct reactions, have the same structural scaffold, the TIM barrel 6., 7.••. The TIM barrel (named after triose phosphate isomerase) comprises eight β-strands and eight α-helices, which are arranged in a cyclically symmetric pattern, with the strands lining the inner core of the barrel. Despite the differences in the catalyzed reactions, the substrate-binding pockets of the three TIM-barrel glycolytic enzymes are located in a similar spatial configuration, in the interior of the barrel lined by the β-strands 6., 7.••, 8.. However, the residues that directly participate in catalysis are different in each of these enzymes. These observations suggested that evolution of enzymes involved not merely changes in substrate specificity, but also considerable exploration of the reaction space. The evolutionary changes involved could range from the use of similar catalytic residues on similar bonds to dramatic variations in the catalytic residues, while retaining the same structural scaffold 9.••, 10.••.

Traditionally, the presence of a characteristic set of conserved residues had been the cornerstone of the identification of enzymes through sequence analysis. However, in cases such as the TIM barrel, where the structure is preserved but catalytic residues vary, detection of evolutionary relationships had been far more difficult. The explosion in the number of structures and sequences in the past few years facilitates the detection of such subtle relationships 7.••, 8.. This allows one to address meaningful general questions regarding the origin and evolution of diverse biochemical activities in related structural contexts. Here, we attempt to use the information from recently reported structures and sequences to provide a perspective on the major principles that seem to shape the emergence of diverse enzymatic activities within structurally similar and evolutionarily related scaffolds.