Characterization of protein–ligand binding interactions of enoyl-ACP reductase (FabI) by native MS reveals allosteric effects of coenzymes and the inhibitor triclosan

Joyner, P. Matthew; Tran, Denise P.; Zenaidee, Muhammad A.; Loo, Joseph A.

doi:10.1002/pro.4252

Characterization of protein–ligand binding interactions of enoyl-ACP reductase (FabI) by native MS reveals allosteric effects of coenzymes and the inhibitor triclosan

Journal Article · Wed Dec 08 23:00:00 EST 2021 · Protein Science

DOI:https://doi.org/10.1002/pro.4252· OSTI ID:1976374

^[1]; Tran, Denise P. ^[2]; Zenaidee, Muhammad A. ^[3]; Loo, Joseph A. ^[4]

Pepperdine University, Malibu, CA (United States); University of California, Los Angeles, CA (United States); OSTI
University of California, Los Angeles, CA (United States); University of Sydney, NSW (Australia)
University of California, Los Angeles, CA (United States); Macquarie University, NSW (Australia)
University of California, Los Angeles, CA (United States)

The enzyme enoyl-ACP reductase (also called FabI in bacteria) is an essential member of the fatty acid synthase II pathway in plants and bacteria. This enzyme is the target of the antibacterial drug triclosan and has been the subject of extensive studies for the past 20 years. Despite the large number of reports describing the biochemistry of this enzyme, there have been no studies that provided direct observation of the protein and its various ligands. In this paper we describe the use of native MS to characterize the protein–ligand interactions of FabI with its coenzymes NAD⁺ and NADH and with the inhibitor triclosan. Measurements of the gas-phase affinities of the enzyme for these ligands yielded values that are in close agreement with solution-phase affinity measurements. Additionally, FabI is a homotetramer and we were able to measure the affinity of each subunit for each coenzyme, which revealed that both coenzymes exhibit a positive homotropic allosteric effect. An allosteric effect was also observed in association with the inhibitor triclosan. These observations provide new insights into this well-studied enzyme and suggest that there may still be gaps in the existing mechanistic models that explain FabI inhibition.

View Accepted Manuscript (DOE)

Research Organization:: University of California, Los Angeles, CA (United States); Pepperdine University, Malibu, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC); National Institutes of Health (NIH)

Grant/Contract Number:: FC02-02ER63421

OSTI ID:: 1976374

Alternate ID(s):: OSTI ID: 1835900

Journal Information:: Protein Science, Journal Name: Protein Science Journal Issue: 3 Vol. 31; ISSN 0961-8368

Publisher:: WileyCopyright Statement

Country of Publication:: United States

Language:: English

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
allosterism
antibacterial
enoyl-ACP reductase
ligand affinity
native MS
protein mass spectrometry
protein-ligand binding
triclosan

Characterization of protein–ligand binding interactions of enoyl-ACP reductase (FabI) by native MS reveals allosteric effects of coenzymes and the inhibitor triclosan

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