Redox Modulation of Oligomeric State in Proline Utilization A

David A. Korasick, Ashley C. Campbell, Shelbi L. Christgen, Srinivas Chakravarthy, Tommi A. White, Donald F. Becker, John J. Tanner

Research output: Contribution to journalArticle

Abstract

Homooligomerization of proline utilization A (PutA) bifunctional flavoenzymes is intimately tied to catalytic function and substrate channeling. PutA from Bradyrhizobium japonicum (BjPutA) is unique among PutAs in that it forms a tetramer in solution. Curiously, a dimeric BjPutA hot spot mutant was previously shown to display wild-type catalytic activity despite lacking the tetrameric structure. These observations raised the question of what is the active oligomeric state of BjPutA. Herein, we investigate the factors that contribute to tetramerization of BjPutA in vitro. Negative-stain electron microscopy indicates that BjPutA is primarily dimeric at nanomolar concentrations, suggesting concentration-dependent tetramerization. Further, sedimentation-velocity analysis of BjPutA at high (micromolar) concentration reveals that although the binding of active-site ligands does not alter oligomeric state, reduction of the flavin adenine dinucleotide cofactor results in dimeric protein. Size-exclusion chromatography coupled with multiangle light scattering and small-angle x-ray scattering analysis also reveals that reduced BjPutA is dimeric. Taken together, these results suggest that the BjPutA oligomeric state is dependent upon both enzyme concentration and the redox state of the flavin cofactor. This is the first report, to our knowledge, of redox-linked oligomerization in the PutA family.

Original languageEnglish (US)
Pages (from-to)2833-2843
Number of pages11
JournalBiophysical journal
Volume114
Issue number12
DOIs
StatePublished - Jun 19 2018

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ASJC Scopus subject areas

  • Biophysics

Cite this

Korasick, D. A., Campbell, A. C., Christgen, S. L., Chakravarthy, S., White, T. A., Becker, D. F., & Tanner, J. J. (2018). Redox Modulation of Oligomeric State in Proline Utilization A. Biophysical journal, 114(12), 2833-2843. https://doi.org/10.1016/j.bpj.2018.04.046