A catalytic dyad modulates conformational change in the CO2-fixing flavoenzyme 2-ketopropyl coenzyme M&nbsp;oxidoreductase/carboxylase

Mattice, Jenna R.; Shisler, Krista A.; DuBois, Jennifer L.; Peters, John W.; Bothner, Brian

doi:10.1016/j.jbc.2022.101884

A catalytic dyad modulates conformational change in the CO₂-fixing flavoenzyme 2-ketopropyl coenzyme M oxidoreductase/carboxylase

Journal Article · Thu Mar 31 00:00:00 EDT 2022 · Journal of Biological Chemistry

DOI:https://doi.org/10.1016/j.jbc.2022.101884· OSTI ID:1904773

Mattice, Jenna R. ^[1]; Shisler, Krista A. ^[2]; DuBois, Jennifer L. ^[3]; Peters, John W. ^[2]; Bothner, Brian ^[3]

Montana State University, Bozeman, MT (United States); Montana State Univ., Bozeman, MT (United States)
Washington State University, Pullman, WA (United States)
Montana State University, Bozeman, MT (United States)

2-Ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a member of the flavin and cysteine disulfide containing oxidoreductase family (DSOR) that catalyzes the unique reaction between atmospheric CO₂ and a ketone/enolate nucleophile to generate acetoacetate. However, the mechanism of this reaction is not well understood. Here, we present evidence that 2-KPCC, in contrast to the well-characterized DSOR enzyme glutathione reductase, undergoes conformational changes during catalysis. Using a suite of biophysical techniques including limited proteolysis, differential scanning fluorimetry, and native mass spectrometry in the presence of substrates and inhibitors, we observed conformational differences between different ligand-bound 2-KPCC species within the catalytic cycle. Analysis of site-specific amino acid variants indicated that 2-KPCC-defining residues, Phe501-His506, within the active site are important for transducing these ligand induced conformational changes. We propose that these conformational changes promote substrate discrimination between H⁺ and CO₂ to favor the metabolically preferred carboxylation product, acetoacetate.

View Accepted Manuscript (DOE)

Research Organization:: Montana State University, Bozeman, MT (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); US Department of Agriculture (USDA); National Institutes of Health (NIH); MSU Office of Research, Economic Development and Graduate Education

Grant/Contract Number:: FG02-04ER15563

OSTI ID:: 1904773

Journal Information:: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 5 Vol. 298; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
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A catalytic dyad modulates conformational change in the CO2-fixing flavoenzyme 2-ketopropyl coenzyme M oxidoreductase/carboxylase

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A catalytic dyad modulates conformational change in the CO₂-fixing flavoenzyme 2-ketopropyl coenzyme M oxidoreductase/carboxylase