Abstract
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 C1–9C29 and N10–C19 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. Understanding the different active site conformations that appear during the catalytic cycle may allow fine-tuning of inhibitor discovery efforts.
Recommended Citation
A. C. Campbell et al., "Trapping Conformational States of a Flavin-dependent N-monooxygenase in Crystallo Reveals Protein and Flavin Dynamics," Journal of Biological Chemistry, vol. 295, no. 38, pp. 13239 - 13250, Elsevier; American Society for Biochemistry and Molecular Biology, Sep 2020.
The definitive version is available at https://doi.org/10.1074/jbc.ra120.014750
Department(s)
Chemistry
Publication Status
Open Access
International Standard Serial Number (ISSN)
1083-351X; 0021-9258
Document Type
Article - Journal
Document Version
Final Version
File Type
text
Language(s)
English
Rights
© 2024 Elsevier; American Society for Biochemistry and Molecular Biology, All rights reserved.
Creative Commons Licensing
This work is licensed under a Creative Commons Attribution 4.0 License.
Publication Date
18 Sep 2020
PubMed ID
32723870
Comments
National Science Foundation, Grant S10 RR029205