7jvk

Structure of the M101A variant of the SidA ornithine hydroxylase with the FAD in the "out" conformation

spinqualitylabelsall models 7jvk, resolution 2.20Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 7jvk is a 4 chain structure with sequence from Aspergillus fumigatus Af293. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 2.2Å Ligands: ACT, CA, FAD, PGE Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function SIDA_ASPFU Catalyzes the conversion of L-ornithine to N(5)-hydroxyornithine, the first step in the biosynthesis of all hydroxamate-containing siderophores, such as the secreted triacetylfusarinine C (TAFC) involved in iron uptake and the intracellular iron storage compound desferriferricrocin (DFFC). Highly specific for its substrate, only hydrolyzing l-ornithine. Has preference for NADPH over NADH, NADPH playing a role in stabilization of the C4a-hydroperoxyflavin intermediate. Essential for virulence. Publication Abstract from PubMed 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. Structural Determinants of Flavin Dynamics in a Class B Monooxygenase.,Campbell AC, Robinson R, Mena-Aguilar D, Sobrado P, Tanner JJ Biochemistry. 2020 Dec 8;59(48):4609-4616. doi: 10.1021/acs.biochem.0c00783. Epub, 2020 Nov 23. PMID:33226785 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Monooxygenase 3D structures

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