The Dodecin from Thermus thermophilus, a Bifunctional Cofactor Storage Protein

Abstract

Dodecins are so far the smallest known flavoproteins (68 -71 amino acids) and are most likely involved in prokaryotic flavin storage. The dodecin monomers adopt a simple ␤␣␤␤-fold and assemble to hollow sphere-like dodecameric complexes. Flavin binding by the dodecin from Thermus thermophilus showed a 1:1 stoichiometry and apparent dissociation constants in the submicromolar to nanomolar range as characterized by isothermal titration calorimetry and fluorescence titrations. The x-ray structures of the flavin-prebound and FMN-reconstituted state of the T. thermophilus dodecin revealed binding of FMN dimers in a novel si-si-rather than the re-re-orientation of their isoalloxazine moieties as found before in an archaeal dodecin. Electron paramagnetic resonance studies demonstrated that upon reduction the excess electron is localized only on one flavin, thus making dodecin-bound flavins highly refractory to redox chemistry. Besides FMN dimers, trimers of coenzyme A are additionally bound to this eubacterial dodecin along the 3-fold symmetry face II of the dodecin complex. Therefore, dodecins can act as bifunctional cofactor storage proteins that sequester catalytic cofactors in prokaryotes very efficiently in an aggregated and unreactive state.Flavoproteins are ubiquitous proteins using flavins as prosthetic groups. Because riboflavin (vitamin B 2 ) serves mostly as a biosynthetic intermediate, FMN and FAD are the principal flavin cofactors (1). They are involved in the catalysis of a wide range of biological redox reactions, such as the dehydrogenation of NAD(P)H, lipid esters, and D-amino acids, the oxidation of amines to imines, the formation and cleavage of disulfide bonds, the hydroxylation of aromatic substrates, or the activation of molecular oxygen. Furthermore, flavoproteins serve as electron transmitters in electron transfer processes like oxidative phosphorylation. Here, they act as mediators between typical 2-electron donors like NADH and 1-electron acceptors like the heme group. This versatility to engage in one-and twoelectron transfer reactions is due to their isoalloxazine moiety that adopts either an oxidized, a semiquinoid, or fully reduced redox state (2). Flavins are not only involved in redox reactions but also in the sensing of blue or ultraviolet light (3). In cryptochromes, flavin chromophores mediate the flowering and daily light/dark cycles in plants (4), in phototropins they regulate phototropism (5), and in photolyases they are involved in DNA repair (6). Beside catalytic or sensoric actions, flavoproteins are also involved in the binding or transport of flavins. During pregnancy, a specific carrier system evolves in vertebrates including the riboflavin-binding proteins (RCPs or RfBPs), since adequate amounts of vitamin B 2 are essential for the normal fetal development (7).Dodecins are a novel family of flavin-binding proteins of unknown function that were discovered in the Archaea Halobacterium salinarum during an inverse structural genomics project on halophilic proteins (8). So far it...

Ultrafast Excited-state Deactivation of Flavins Bound to Dodecin

“…The fastest time constant 1 , close to the instrumental response time, is necessary to describe the rise of the signal. Another time constant, 4 , longer than the maximum delay time of the experiment, is used to include residual signals, which are here nearly 0. Two further time constants, 2 and 3 , describe the decay of the difference signals: a fast time constant 2 of 800 Ϯ 100 fs and a slower one 3 of 4 Ϯ 1 ps.…”

“…With one ligand per 68 residues, dodecin represents the flavoprotein with highest flavin load known to date. Dodecin has also been found in bacteria where it was characterized as a binder of FMN (2)(3)(4).…”

“…Structures revealed a dodecameric fold, providing six identical binding pockets, which each enables binding of two antiparallel arranged flavins between two tryptophan residues building an aromatic tetrade arrangement ( Fig. 1A) (1)(2)(3)(4)(5)(6)(7).…”

Crystal structure of calcium dodecin (Rv0379), from Mycobacterium tuberculosis with a unique calcium‐binding site

“…The overall three-dimensional architecture of the Ca dodecin oligomer is similar to the Halobacterium salinarum dodecin (hsDodecin), a riboflavinbinding protein involved in riboflavin homeostasis. [14][15][16][17] The three subunits bind one calcium ion at the interface [ Fig. 1(b)], and the association of four equivalent sets of three subunits and a calcium leads to dodecameric oligomerization [ Fig.…”

Reverse Structural Genomics

“…A related flavin binding mode was recently discovered in a family of archaeal and bacterial proteins. These so-called dodecins consist of 12 protomers of 68 -71 amino acids with six flavin-binding sites (23). As in ppBat, two tryptophans sandwich the isoalloxazine or alloxazine (lumichrome) ring system, although each binding site accommodates two ligands instead of one.…”