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

Fingerprint

Proline
Oxidation-Reduction
Bradyrhizobium
Flavin-Adenine Dinucleotide
Gel Chromatography
Catalytic Domain
Electron Microscopy
Coloring Agents
Binding Sites
X-Rays
Ligands
Light
Enzymes
Proteins

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

Redox Modulation of Oligomeric State in Proline Utilization A. / Korasick, David A.; Campbell, Ashley C.; Christgen, Shelbi L.; Chakravarthy, Srinivas; White, Tommi A.; Becker, Donald F; Tanner, John J.

In: Biophysical journal, Vol. 114, No. 12, 19.06.2018, p. 2833-2843.

Research output: Contribution to journalArticle

Korasick, DA, Campbell, AC, Christgen, SL, Chakravarthy, S, White, TA, Becker, DF & Tanner, JJ 2018, 'Redox Modulation of Oligomeric State in Proline Utilization A', Biophysical journal, vol. 114, no. 12, pp. 2833-2843. https://doi.org/10.1016/j.bpj.2018.04.046
Korasick DA, Campbell AC, Christgen SL, Chakravarthy S, White TA, Becker DF et al. Redox Modulation of Oligomeric State in Proline Utilization A. Biophysical journal. 2018 Jun 19;114(12):2833-2843. https://doi.org/10.1016/j.bpj.2018.04.046
Korasick, David A. ; Campbell, Ashley C. ; Christgen, Shelbi L. ; Chakravarthy, Srinivas ; White, Tommi A. ; Becker, Donald F ; Tanner, John J. / Redox Modulation of Oligomeric State in Proline Utilization A. In: Biophysical journal. 2018 ; Vol. 114, No. 12. pp. 2833-2843.
@article{b55cf47e42f44ae0a7451530a5fe173e,
title = "Redox Modulation of Oligomeric State in Proline Utilization A",
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.",
author = "Korasick, {David A.} and Campbell, {Ashley C.} and Christgen, {Shelbi L.} and Srinivas Chakravarthy and White, {Tommi A.} and Becker, {Donald F} and Tanner, {John J.}",
year = "2018",
month = "6",
day = "19",
doi = "10.1016/j.bpj.2018.04.046",
language = "English (US)",
volume = "114",
pages = "2833--2843",
journal = "Biophysical Journal",
issn = "0006-3495",
publisher = "Biophysical Society",
number = "12",

}

TY - JOUR

T1 - Redox Modulation of Oligomeric State in Proline Utilization A

AU - Korasick, David A.

AU - Campbell, Ashley C.

AU - Christgen, Shelbi L.

AU - Chakravarthy, Srinivas

AU - White, Tommi A.

AU - Becker, Donald F

AU - Tanner, John J.

PY - 2018/6/19

Y1 - 2018/6/19

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=85048639040&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85048639040&partnerID=8YFLogxK

U2 - 10.1016/j.bpj.2018.04.046

DO - 10.1016/j.bpj.2018.04.046

M3 - Article

VL - 114

SP - 2833

EP - 2843

JO - Biophysical Journal

JF - Biophysical Journal

SN - 0006-3495

IS - 12

ER -