Trapping conformational states of a flavin-dependent N-monooxygenase in crystallo reveals protein and flavin dynamics

Campbell, Ashley C.; Stiers, Kyle M.; Martin Del Campo, Julia S.; Mehra-Chaudhary, Ritcha; Sobrado, Pablo; Tanner, John J.

doi:10.1074/jbc.ra120.014750

Title: Trapping conformational states of a flavin-dependent N-monooxygenase in crystallo reveals protein and flavin dynamics

Journal Article · Tue Jul 28 00:00:00 EDT 2020 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.ra120.014750· OSTI ID:1673780

^[1]; Stiers, Kyle M. ^[1]; Martin Del Campo, Julia S. ^[2]; Mehra-Chaudhary, Ritcha ^[1];

^[2];

^[1]

Univ. of Missouri, Columbia, MO
Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States)

The siderophore biosynthetic enzyme A (SidA) ornithine hydroxylase from Aspergillus fumigatus is a fungal disease drug target involved in the production of hydroxamate-containing siderophores, which are used by the pathogen to sequester iron. SidA is an N-monooxygenase that catalyzes the NADPH-dependent hydroxylation of l-ornithine through a multistep oxidative mechanism, utilizing a C4a-hydroperoxyflavin intermediate. Here we present four new crystal structures of SidA in various redox and ligation states, including the first structure of oxidized SidA without NADP(H) or l-ornithine bound (resting state). The resting state structure reveals a new out active site conformation characterized by large rotations of the FAD isoalloxazine around the C1–'C2' and N10–C1' bonds, coupled to a 10-Å movement of the Tyr-loop. Additional structures show that either flavin reduction or the binding of NADP(H) is sufficient to drive the FAD to the in conformation. The structures also reveal protein conformational changes associated with the binding of NADP(H) and l-ornithine. Some of these residues were probed using site-directed mutagenesis. Docking was used to explore the active site of the out conformation. These calculations identified two potential ligand-binding sites. Altogether, our results provide new information about conformational dynamics in flavin-dependent monooxygenases. Overall, understanding the different active site conformations that appear during the catalytic cycle may allow fine-tuning of inhibitor discovery efforts.

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Research Organization:: Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

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

Grant/Contract Number:: AC02-05CH11231; AC02-06CH11357; P30 GM124165; S10 RR029205; S10OD021527

OSTI ID:: 1673780

Journal Information:: Journal of Biological Chemistry, Vol. 295, Issue 38; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
X-ray crystallography
enzyme structure
flavoprotein
enzyme kinetics
site-directed mutagenesis
siderophore
flavin-dependent monooxygenases
ornithine hydroxylase

Title: Trapping conformational states of a flavin-dependent N-monooxygenase in crystallo reveals protein and flavin dynamics

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