Valentino, HannahSobrado, Pablo2022-01-132022-01-132021-09-280006-2960http://hdl.handle.net/10919/107580N-hydroxylating monooxygenases (NMOs) are a subclass of flavin-dependent enzymes that hydroxylate nitrogen atoms. Recently, unique NMOs that perform multiple reactions on one substrate molecule have been identified. Fosfazinomycin M (FzmM) is one such NMO, forming nitrosuccinate from aspartate (Asp) in the fosfazinomycin biosynthetic pathway in someStreptomycessp. This work details the biochemical and kinetic analysis of FzmM. Steady-state kinetic investigation shows that FzmM performs a coupled reaction with Asp (kcat, 3.0 ± 0.01 s-1) forming nitrosuccinate, which can be converted to fumarate and nitrite by the action of FzmL. FzmM displays a 70-fold higherkcat/KMvalue for NADPH compared to NADH and has a narrow optimal pH range (7.5-8.0). Contrary to other NMOs where thekredis rate-limiting, FzmM exhibits a very fastkred(50 ± 0.01 s-1at 4 °C) with NADPH. NADPH binds at aKDvalue of ∼400 μM, and hydride transfer occurs withpro-Rstereochemistry. Oxidation of FzmM in the absence of Asp exhibits a spectrum with a shoulder at ∼370 nm, consistent with the formation of a C(4a)-hydroperoxyflavin intermediate, which decays into oxidized flavin and hydrogen peroxide at a rate 100-fold slower than thekcat. This reaction is enhanced in the presence of Asp with a slightly fasterkoxthan thekcat, suggesting that flavin dehydration or Asp oxidation is partially rate limiting. Multiple sequence analyses of FzmM to NMOs identified conserved residues involved in flavin binding but not for NADPH. Additional sequence analysis to related monooxygenases suggests that FzmM shares sequence motifs absent in other NMOs.Pages 2851-286414 page(s)application/pdfenIn CopyrightLife Sciences & BiomedicineBiochemistry & Molecular BiologyORNITHINE-HYDROXYLASESTRUCTURAL DETERMINANTSPSEUDOMONAS-AERUGINOSAKINETIC MECHANISMOXYGEN ACTIVATIONFLAVINMONOOXYGENASESUBSTRATEIDENTIFICATIONSELECTIVITY0304 Medicinal and Biomolecular Chemistry0601 Biochemistry and Cell Biology1101 Medical Biochemistry and MetabolomicsSuccinic AcidHydrazinesDinitrocresolsOrganophosphorus CompoundsFlavin-Adenine DinucleotideNADPMixed Function OxygenasesFlavinsOxidation-ReductionHydroxylationKineticsArticle - Refereed2022-01-13https://doi.org/10.1021/acs.biochem.1c005126038Sobrado, Pablo [0000-0003-1494-5382]345161021520-4995