Year: 2021
Type: article
Abstract: N -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 some Streptomyces sp. This work details the biochemical and kinetic analysis of FzmM. Steady-state kinetic investigation shows that FzmM performs a coupled reaction with Asp ( k cat, 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 higher k cat / K M value for NADPH compared to NADH and has a narrow optimal pH range (7.5–8.0). Contrary to other NMOs where the k red is rate-limiting, FzmM exhibits a very fast k red (50 ± 0.01 s –1 at 4 °C) with NADPH. NADPH binds at a K D value of ∼400 μM, and hydride transfer occurs with pro-R stereochemistry. 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 the k cat . This reaction is enhanced in the presence of Asp with a slightly faster k ox than the k cat, 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.
Source: Biochemistry
Authors: Hannah Valentino, Pablo Sobrado
Institution: Virginia Tech
Language: English
Subfield: Pharmacology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W3201534805