Defining structural features of the family of tetrameric flavoproteins WrbA

 J. Wolfová1, J. Carey2, J. Brynda1,3, R. Ettrich1,4, I. Kutá Smatanová1,4


1Institute of Physical Biology, University of South Bohemia České Budějovice, Zámek 136,
CZ-373 33 Nové Hrady, Czech Republic

2Chemistry Department, Princeton University, Washington Rd and William St, Princeton, NJ 08544-1009, USA

3Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, CZ-16637 Prague 6, Czech Republic

                4Institute of Systems Biology and Ecology, Academy of Science of the Czech Republic, Zámek 136,CZ-373 33 Nové Hrady, Czech Republic


Flavoproteins WrbA form a distinct family of flavodoxin-like proteins that is widely distributed in living organisms, from bacteria to fungi and higher plants [1, 2]. These proteins were identified as tetrameric NADH:quinone oxidoreductases (E.C. [2], carrying out two-electron transport from NADH to quinones by using flavin mononucleotide (FMN) as the redox-active cofactor [2, 3, 4]. Together with other enzymes performing the two-electron reductions of quinones [5, 6, 7], members of the WrbA family are thought to participate in the cell protection against oxidative stress. Structural investigations of the prototypical WrbA from Escherichia coli and its homologues in other bacteria [8, 9] confirmed the previous observations and sequentially-based predictions that in each structure four WrbA monomers form a tetramer, where individual subunits share the common fold of the related flavodoxins [10] with sequence insertions unique for WrbA family forming additional secondary structure elements. Unlike typically monomeric flavodoxins that are involved in one-electron transport processes between protein partners, tetrameric flavoproteins WrbA execute the two-electron reductions of quinones. To understand these functional distinctions and the unique tetramerization ability of WrbA in context with the molecular structure and to find the defining structural features of the WrbA family, the detailed comparative structural analysis of E. coli WrbA with the related flavodoxins and the functionally homologous eukaryotic FAD-dependent quinone oxidoreductase was performed.

Structural analysis included three structural models of E. coli WrbA obtained by our group using X-ray diffraction on single crystals [11]: two crystal structures of the protein complexed with FMN (holoWrbA, two crystal forms, PDB IDs: 2R96 and 2R97), one crystal structure of the protein without FMN bound (apoWrbA, PDB ID: 2RG1). The structures were compared by the 3D-superposition with long-chain holo- and apoflavodoxin from Anabaena (PDB IDs: 1FLV and 1FTG, respectively; [12, 13]) and with mammalian NAD(P)H:quinone oxidoreductase (Nqo, PDB ID: 1QRD; [7]).

Structural comparison of the monomers of holoWrbA and holoflavodoxin revealed only one characteristic sequence insertion distinguishing WrbA from flavodoxins, in contrast with previous reports. Structurally the unique insertions form small subdomains contacting each other at the ‘poles’ of the WrbA tetramers. Nevertheless, analysis of interfaces of the WrbA tetramers indicated that the key elements promoting tetramerization correspond to the integral secondary structure elements of the flavodoxin fold and thus the unique subdomain of WrbA is not dedicated to tetramerization as earlier proposed. Tetramer appears to be the obligate functional assembly of WrbA, with residues of the three subunits participating on the formation of the FMN-binding site. Comparison of the WrbA FMN-binding site with those of flavodoxin and Nqo showed that tetrameric WrbA forms a cavernous active site similar to that of dimeric Nqo, that analogously to WrbA promotes two-electron reduction of the electrophilic substrates. Assembly of the flavodoxin-like fold into tetramers to form active site of WrbA seems to be adaptation to the two-electron redox reaction. Despite the different FMN-binding sites of WrbA and flavodoxin striking similarities were observed in the behavior of the FMN-binding residues in response to FMN binding.

The detailed comparative study of WrbA structures enabled specification of the defining structural features of the WrbA family and sharpening of the view of the relationship between WrbA, flavodoxins and eukaryotic quinone oxidoreductases. Suprising finding of unifying features with the related protein families indicates WrbA to be a significant member of flavodoxin-like proteins. 


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This work was supported by the Ministry of Education of the Czech Republic (Kontakt ME09016, MSM6007665808, LC06010, by the Academy of Sciences of the Czech Republic (AV0Z60870520) and by the U.S. National Science Foundation (INT03-09049 to J.C.)