Abstract
Allicin is a component of the characteristic smell and flavor of garlic (Allium sativum). A flavin-containing monooxygenase (FMO) produced by A. sativum (AsFMO) was previously proposed to oxidize S-allyl-L-cysteine (SAC) to alliin, an allicin precursor. Here, we present a kinetic and structural characterization of AsFMO that suggests a possible contradiction to this proposal. Results of steady-state kinetic analyses revealed that AsFMO exhibited negligible activity with SAC; however, the enzyme was highly active with L-cysteine, N-acetyl-L-cysteine, and allyl mercaptan. We found that allyl mercaptan with NADPH was the preferred substrate-cofactor combination. Rapid-reaction kinetic analyses showed that NADPH binds tightly (KD of ~2 mM) to AsFMO and that the hydride transfer occurs with pro-R stereospecificity. We detected the formation of a long-wavelength band when AsFMO was reduced by NADPH, probably representing the formation of a charge-transfer complex. In the absence of substrate, the reduced enzyme, in complex with NADP1, reacted with oxygen and formed an intermediate with a spectrum characteristic of C4a-hydroperoxyflavin, which decays several orders of magnitude more slowly than the kcat. The presence of substrate enhanced C4a-hydroperoxyflavin formation and, upon hydroxylation, oxidation occurred with a rate constant similar to the kcat. The structure of AsFMO complexed with FAD at 2.08-Å resolution features two domains for binding of FAD and NADPH, representative of class B flavin monooxygenases. These biochemical and structural results are consistent with AsFMO being an S-monooxygenase involved in allicin biosynthesis through direct formation of sulfenic acid and not SAC oxidation.
Recommended Citation
H. Valentino et al., "Structure and Function of a Flavin-dependent S-monooxygenase from Garlic (Allium Sativum)," Journal of Biological Chemistry, vol. 295, no. 32, pp. 11042 - 11055, Elsevier; American Society for Biochemistry and Molecular Biology, Aug 2020.
The definitive version is available at https://doi.org/10.1074/jbc.ra120.014484
Department(s)
Chemistry
Publication Status
Open Access
International Standard Serial Number (ISSN)
1083-351X; 0021-9258
Document Type
Article - Journal
Document Version
Final Version
File Type
text
Language(s)
English
Rights
© 2024 Elsevier; American Society for Biochemistry and Molecular Biology, All rights reserved.
Creative Commons Licensing
This work is licensed under a Creative Commons Attribution 4.0 License.
Publication Date
07 Aug 2020
PubMed ID
32527723
Comments
National Science Foundation, Grant CHE 2003658