Abstract
Structural and biochemical constraints force some segments of proteins to evolve more slowly than others, often allowing identification of conserved structural or sequence motifs that can be associated with substrate binding properties, chemical mechanisms, and molecular functions. We have assessed the functional and structural constraints imposed by cofactors on the evolution of new functions in a superfamily of flavoproteins characterized by two-dinucleotide binding domains, the “two dinucleotide binding domains” flavoproteins (tDBDF) superfamily. Although these enzymes catalyze many different types of oxidation/reduction reactions, each is initiated by a stereospecific hydride transfer reaction between two cofactors, a pyridine nucleotide and flavin adenine dinucleotide (FAD). Sequence and structural analysis of more than 1,600 members of the superfamily reveals new members and identifies details of the evolutionary connections among them. Our analysis shows that in all of the highly divergent families within the superfamily, these cofactors adopt a conserved configuration optimal for stereospecific hydride transfer that is stabilized by specific interactions with amino acids from several motifs distributed among both dinucleotide binding domains. The conservation of cofactor configuration in the active site restricts the pyridine nucleotide to interact with FAD from the re-side, limiting the flow of electrons from the re-side to the si-side. This directionality of electron flow constrains interactions with the different partner proteins of different families to occur on the same face of the cofactor binding domains. As a result, superimposing the structures of tDBDFs aligns not only these interacting proteins, but also their constituent electron acceptors, including heme and iron-sulfur clusters. Thus, not only are specific aspects of the cofactor-directed chemical mechanism conserved across the superfamily, the constraints they impose are manifested in the mode of protein–protein interactions. Overlaid on this foundation of conserved interactions, nature has conscripted different protein partners to serve as electron acceptors, thereby generating diversification of function across the superfamily.
Highlights
The large disparity between the number of unique protein folds and the number of unique proteins that exist in biological organisms [1] indicates that nature has utilized a relatively small number of folds to generate a large number of different functions
Evolving a new function often occurs through divergence from a parental gene; constraints associated with a parent scaffold can be linked to a wide array of properties—including folding, cofactor binding, chemical mechanism, or interactions with substrates—that constrain regions of the gene to evolve at slower rates, giving rise to conserved structural features recognizable from sequence or structural comparisons
All two dinucleotide binding domains’’ flavoproteins (tDBDF), as the name implies, have in common two dinucleotide binding Rossmann fold domains fused in a single peptide chain. Both domains are required for function, and both are always present in all the members of the superfamily. Each of these domains binds one of the two dinucleotide cofactors, flavin adenine dinucleotide (FAD) and a pyridine nucleotide, respectively. (A notable exception is the flavocytochrome c sulfide dehydrogenase family, in which the pyridine nucleotide is replaced by hydrogen sulfide.) In most tDBDF superfamily members, the N-terminal domain binds the FAD and the C-terminal domain binds the pyridine nucleotide
Summary
The large disparity between the number of unique protein folds and the number of unique proteins that exist in biological organisms [1] indicates that nature has utilized a relatively small number of folds to generate a large number of different functions. Several studies have demonstrated that aspects of chemical mechanism, in particular, can constrain evolution of new functions in enzyme superfamilies [2,3,4,5] Members of such mechanistically diverse superfamilies have evolved to catalyze a wide range of overall reactions using a common partial reaction or chemical attribute (see [6,7] and references therein for examples). These partial reactions are mediated by highly conserved structural features in the active site.
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