Characterization of a Nitro-Forming Enzyme Involved in Fosfazinomycin Biosynthesis.
Biochemistry
; 60(38): 2851-2864, 2021 09 28.
Article
em En
| MEDLINE
| ID: mdl-34516102
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 (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 higher kcat/KM value for NADPH compared to NADH and has a narrow optimal pH range (7.5-8.0). Contrary to other NMOs where the kred is rate-limiting, FzmM exhibits a very fast kred (50 ± 0.01 s-1 at 4 °C) with NADPH. NADPH binds at a KD value of â¼400 µM, and hydride transfer occurs with pro-R stereochemistry. Oxidation of FzmM in the absence of Asp exhibits a spectrum with a shoulder at â¼370 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 kcat. This reaction is enhanced in the presence of Asp with a slightly faster kox than the kcat, 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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Compostos Organofosforados
/
Hidrazinas
Idioma:
En
Revista:
Biochemistry
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Estados Unidos