Browsing by Author "Campbell, Ashley C."

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    Structural Determinants of Flavin Dynamics in a Class B Monooxygenase
    Campbell, Ashley C.; Robinson, Reeder; Mena-Aguilar, Didier; Sobrado, Pablo; Tanner, John J. (American Chemical Society, 2020-12-08)
    The ornithine hydroxylase known as SidA is a class B flavin monooxygenase that catalyzes the first step in the biosynthesis of hydroxamate-containing siderophores in Aspergillus fumigatus. Crystallographic studies of SidA revealed that the FAD undergoes dramatic conformational changes between out and in states during the catalytic cycle. We sought insight into the origins and purpose of flavin motion in class B monooxygenases by probing the function of Met101, a residue that contacts the pyrimidine ring of the in FAD. Steady-state kinetic measurements showed that the mutant variant M101A has a 25-fold lower turnover number. Pre-steady-state kinetic measurements, pH profiles, and solvent kinetic isotope effect measurements were used to isolate the microscopic step that is responsible for the reduced steady-state activity. The data are consistent with a bottleneck in the final step of the mechanism, which involves flavin dehydration and the release of hydroxy-l-ornithine and NADP+. Crystal structures were determined for M101A in the resting state and complexed with NADP+. The resting enzyme structure is similar to that of wild-type SidA, consistent with M101A exhibiting normal kinetics for flavin reduction by NADPH and wild-type affinity for NADPH. In contrast, the structure of the M101A-NADP+ complex unexpectedly shows the FAD adopting the out conformation and may represent a stalled conformation that is responsible for the slow kinetics. Altogether, our data support a previous proposal that one purpose of the FAD conformational change from in to out in class B flavin monooxygenases is to eject spent NADP+ in preparation for a new catalytic cycle.
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    Trapping conformational states of a flavin-dependent N-monooxygenase in crystallo reveals protein and flavin dynamics
    Campbell, Ashley C.; Stiers, Kyle M.; Del Campo, Julia S. Martin; Mehra-Chaudhary, Ritcha; Sobrado, Pablo; Tanner, John J. (2020-09-18)
    The siderophore biosynthetic enzyme A (SidA) ornithine hydroxylase fromAspergillus fumigatusis a fungal disease drug target involved in the production of hydroxamate-containing siderophores, which are used by the pathogen to sequester iron. SidA is anN-monooxygenase that catalyzes the NADPH-dependent hydroxylation ofl-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) orl-ornithine bound (resting state). The resting state structure reveals a newoutactive site conformation characterized by large rotations of the FAD isoalloxazine around the C1-' C2 ' and N10-C1 ' bonds, coupled to a 10-angstrom 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 theinconformation. The structures also reveal protein conformational changes associated with the binding of NADP(H) andl-ornithine. Some of these residues were probed using site-directed mutagenesis. Docking was used to explore the active site of theoutconformation. 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.