Flavin Storage and Sequestration by Mycobacterium tuberculosis Dodecin

Abstract

Dodecins are small flavin binding proteins occurring in archaea and bacteria. They are remarkable for binding dimers of flavins with their functional relevant aromatic isoalloxazine rings deeply covered. Bacterial dodecins are widely spread and found in a large variety of pathogens, among them Pseudomonas aeruginosa, Streptococcus pneumonia, Ralstonia solanacearum, and Mycobacterium tuberculosis ( M. tuberculosis). In this work, we seek to understand the function of dodecins from M. tuberculosis dodecin. We describe flavin binding in thermodynamic and kinetic properties and achieve mechanistic insight in dodecin function by applying spectroscopic and electrochemical methods. Intriguingly, we reveal a significant pH dependence in the affinity and specificity of flavin binding. Our data give insight in M. tuberculosis dodecin function and advance the current understanding of dodecins as flavin storage and sequestering proteins. We suggest that the dodecin in M. tuberculosis may specifically be important for flavin homeostasis during the elaborate lifestyle of this organism, which calls for the evaluation of this protein as drug target.

Dodecin as carrier protein for immunizations and bioengineering applications

“…Cells were grown in terrific broth (TB) medium to an optical density at 600 nm (OD 600 ) of about 0.6-0.8 at 37 °C before induction with isopropyl-β-D-thiogalactopyranosid (IPTG; 0.5 mM final concentration), and expression was performed over night at 20 °C. Since mtDod is a flavin binding protein (preferred flavin-ligand is riboflavin-5'-phosphate (FMN)), 19,20 its overexpression causes increased amounts of cellular flavin, leading to a yellowish coloring of the cells. Cells were lysed by French press, and the cell debris was removed by centrifugation.…”

“…5 and ionic strength >100 mM, e.g., in PBS), and the thermal stability can be further increased by adding the native FMN ligand in excess. 19,20 Depending on the stability of the fused cargo, lower temperatures during the heat denaturation may be necessary, or different purification approaches need to be applied (affinity chromatography). For example, mtDod-mmACP started to precipitate at about 55-60 °C in spite of mtDod staying intact, as indicated by maintained FMN binding and preserved dodecameric stability (Fig.…”

“…MtDod constructs that aggregate in inclusion bodies can be refolded by dialysis, as previously described 19 , under conditions optimized for the respective fused cargo. All inclusion bodies were first washed and then dissolved by denaturation using 6 M guanidinium chloride.…”

“…We have recently established the cyclic thermal shift assay, termed thermocyclic fluorescence assay, to determine the stability of dodecins. 19 This assay is based on the fluorescence quenching that is observed when flavins bind to dodecin. In each binding pocket of the dodecamer, the two isoalloxazine ring systems of two bound flavins are embedded between symmetry-related tryptophans.…”

“…[3][4][5] The dodecin protein family was recently discovered as a flavin storage and buffering system that occurs in bacteria and archaea, but not in eukaryotes. [16][17][18][19] Dodecins are 8 kDa small proteins of βαββ-topology. Although forming a small antiparallel β-sheet that partly enwraps the helix, the dodecin fold is unique.…”