Structural Determinants of Flavin Dynamics in a Class B Monooxygenase

Abstract

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 <i>Aspergillus fumigatus</i>. Crystallographic studies of SidA revealed that the FAD undergoes dramatic conformational changes between <i>out</i> and <i>in</i> 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 <i>in</i> 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 <i>out</i> 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 <i>in</i> to <i>out</i> in class B flavin monooxygenases is to eject spent NADP⁺ in preparation for a new catalytic cycle.