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Unprecedented pathway of reducing equivalents in a diflavin-linked disulfide oxidoreductase

  1. Monica Balserab,1
  1. aMetabolic Engineering Group, Departamento de Microbiología y Genética, Universidad de Salamanca, 37007 Salamanca, Spain;
  2. bInstituto de Recursos Naturales y Agrobiología de Salamanca, Consejo Superior de Investigaciones Científicas (CSIC), 37008 Salamanca, Spain;
  3. cInstituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, 41092 Sevilla, Spain;
  4. dInstitute of Biocomputation and Physics of Complex Systems (BIFI), Universidad de Zaragoza, 50009 Zaragoza, Spain;
  5. eJoint Units Instituto de Química-Física Rocasolano (IQFR)-CSIC-BIFI and Grupo de Bioquímica, Biofísica y Biología Computacional (GBsC)-CSIC-BIFI, Universidad de Zaragoza, 50009 Zaragoza, Spain;
  6. fDepartment of Biochemistry and Molecular and Cell Biology, Universidad de Zaragoza, 50009 Zaragoza, Spain;
  7. gAragon Institute for Health Research, 50009 Zaragoza, Spain;
  8. hBiomedical Research Networking Centre for Liver and Digestive Diseases, 28029 Madrid, Spain;
  9. iFundación Aragonesa para la Investigación y Desarrollo, 50018 Zaragoza, Spain;
  10. jInstituto de Biología Molecular y Celular del Cáncer, CSIC-Universidad de Salamanca, 37007 Salamanca, Spain;
  11. kLaboratoire de Biologie Moléculaire et Cellulaire, Université de Neuchatel, 2000 Neuchatel, Switzerland;
  12. lDepartment of Plant and Microbial Biology, University of California, 94720 Berkeley
  1. Contributed by Bob B. Buchanan, October 6, 2017 (sent for review August 3, 2017; reviewed by Juan A. Hermoso and Charles H. Williams)

Significance

Diflavin-linked disulfide oxidoreductases (DDORs) are structurally related to the low-molecular-weight type NADP-dependent thioredoxin reductases, although they do not share a common function. The biochemical and structural characterization of DDORs has revealed a previously unreported mechanism for the transfer of reducing equivalents in flavoenzymes. The present work illustrates the extent to which nature has experimented with flavins as enzyme cofactors in the evolution of redox reactions.

Abstract

Flavoproteins participate in a wide variety of physiologically relevant processes that typically involve redox reactions. Within this protein superfamily, there exists a group that is able to transfer reducing equivalents from FAD to a redox-active disulfide bridge, which further reduces disulfide bridges in target proteins to regulate their structure and function. We have identified a previously undescribed type of flavin enzyme that is exclusive to oxygenic photosynthetic prokaryotes and that is based on the primary sequence that had been assigned as an NADPH-dependent thioredoxin reductase (NTR). However, our experimental data show that the protein does not transfer reducing equivalents from flavins to disulfides as in NTRs but functions in the opposite direction. High-resolution structures of the protein from Gloeobacter violaceus and Synechocystis sp. PCC6803 obtained by X-ray crystallography showed two juxtaposed FAD molecules per monomer in redox communication with an active disulfide bridge in a variant of the fold adopted by NTRs. We have tentatively named the flavoprotein “DDOR” (diflavin-linked disulfide oxidoreductase) and propose that its activity is linked to a thiol-based transfer of reducing equivalents in bacterial membranes. These findings expand the structural and mechanistic repertoire of flavoenzymes with oxidoreductase activity and pave the way to explore new protein engineering approaches aimed at designing redox-active proteins for diverse biotechnological applications.

Footnotes

  • ?1To whom correspondence may be addressed. Email: view{at}berkeley.edu or monica.balsera{at}csic.es.
  • Author contributions: R.M.B., P.S., B.B.B., and M.B. designed research; R.M.B., J.B.A., L.L.-M., S.G.-T., A.V.-C., and M.B. performed research; R.M.B., J.L.R., and M.B. contributed new reagents/analytic tools; R.M.B., J.B.A., L.L.-M., A.V.-C., J.M.d.P., F.J.F., P.S., B.B.B., and M.B. analyzed data; and R.M.B., J.B.A., P.S., B.B.B., and M.B. wrote the paper.

  • Reviewers: J.A.H., Instituto de Química-Física Rocasolano, CSIC; and C.H.W., University of Michigan.

  • The authors declare no conflict of interest.

  • Data deposition: The atomic coordinates and structure factors reported in this paper have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID codes 5JRI, 5K0A, 5ODE, and 5N0J).

  • This article contains supporting information online at www.danielhellerman.com/lookup/suppl/doi:10.1073/pnas.1713698114/-/DCSupplemental.

Published under the PNAS license.

Online Impact

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