Evolution of Function in the “Two Dinucleotide Binding Domains” Flavoproteins

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.

A Novel F420-dependent Thioredoxin Reductase Gated by Low Potential FAD

“…This caution aside, the report opens up the long studied flavin-dependent thioredoxin reductase family for further exploration. These enzymes belong to a broader disulfide oxidoreductase family of flavoenzymes (lipoamide dehydrogenase, glutathione reductase, trypanothione reductase, mercuric reductase, and NADH peroxidase), which are known for the diversity of their substrates (46). However, the members that have been studied thus far are nicotinamidedependent, and the possibility that some of the uncharacterized members use other types of electron carriers has not been explored.…”

“…Because of high sequence similarities between flavin-containing thioredoxin reductases and other pyridine dinucleotide oxidoreductase family proteins (46), especially the alkyl hydroperoxide reductases, we employed a two-step method involving the following for the identification of DFTR homologs: identification of candidates via Psi-BLAST searches (47) using Mj-DFTR (locus number, MJ1536) as a query and with stringent parameters (low e-value, 1e Ϫ25 ; 10 iterations); phylogenetic analysis-based screening of the candidates. Such an analysis of the extracted methanogen proteins suggested that within this group the DFTR homologs were limited to the phylogenetically deeply rooted methanogens belonging to the class of Methanococci under the euryarchaeal phylum, and these organisms lacked NTR and ferredoxin-thioredoxin reductase (FTR) homologs ( Fig.…”

Flavin-containing monooxygenases in aging and disease: Emerging roles for ancient enzymes

“…FMOs, BVMOs, and NMOs are each distinguished by variations in primary structural motifs, substrate preferences, and catalytic mechanisms. FMOs are also notably similar to the Group A enzymes dihydrolipoamide dehydrogenase (DLD), glutathione reductase (GR), and low-molecular-weight thioredoxin reductase (TRXR) (2,4). These groups are distinguished from other flavin-dependent monooxygenases (Groups C-H) by their combined use of FAD and NAD(P)H. Any functional interplay between FMOs and Group A enzymes, perhaps as an NAD(P)H-thiol redox buffering system, is largely unexplored (5).…”

Exploring the structure and function paradigm

“…Given the complexity of the relationship between sequence, structure and function divergence in proteins sensu lato, a sensible approach is to study these relationships in the limited context of individual families, superfamilies and structural folds (for several recent examples of such studies, see [19][20][21][22][23]). In fact, functional diversity within a given fold or superfamily can span a wide variety of different molecular activities and biological processes, and studies of large and diverse superfamilies are particularly relevant given the recent evidence that they are more likely to contain proteins with essential functions [24].…”

“…Divergence of function amongst homologues can result from several mechanisms such as substitutions in the active sites [27], changes in residues that determine the specificity of interactions (see [28;29] for recent methods that exploit this principle), or variation in environmental context such as the presence of other potential protein interaction partners [23]. However, one conclusion derived from the analysis of the data in the SFLD is that many functionally divergent enzyme superfamilies seem to conserve a common partial reaction or other chemical capability [26].…”