Structural and\nFunctional Characterization of a Novel\nClass A Flavin Monooxygenase from <i>Bacillus niacini</i>

A gene cluster responsible for the degradation of nicotinic\nacid\n(NA) in <i>Bacillus niacini</i> has recently been identified,\nand the structures and functions of the resulting enzymes are currently\nbeing evaluated to establish pathway intermediates. One of the genes\nwithin this cluster encodes a flavin monooxygenase (BnFMO) that is\nhypothesized to catalyze a hydroxylation reaction. Kinetic analyses\nof the recombinantly purified BnFMO suggest that this enzyme catalyzes\nthe hydroxylation of 2,6-dihydroxynicotinic acid (2,6-DHNA) or 2,6-dihydroxypyridine\n(2,6-DHP), which is formed spontaneously by the decarboxylation of\n2,6-DHNA. To understand the details of this hydroxylation reaction,\nwe determined the structure of BnFMO using a multimodel approach combining\nprotein X-ray crystallography and cryo-electron microscopy (cryo-EM).\nA liganded BnFMO cryo-EM structure was obtained in the presence of\n2,6-DHP, allowing us to make predictions about potential catalytic\nresidues. The structural data demonstrate that BnFMO is trimeric,\nwhich is unusual for Class A flavin monooxygenases. In both the electron\ndensity and coulomb potential maps, a region at the trimeric interface\nwas observed that was consistent with and modeled as lipid molecules.\nHigh-resolution mass spectral analysis suggests that there is a mixture\nof phosphatidylethanolamine and phosphatidylglycerol lipids present.\nTogether, these data provide insights into the molecular details of\nthe central hydroxylation reaction unique to the aerobic degradation\nof NA in <i>Bacillus niacini</i>.