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Structural and chemical trapping of flavin‐oxide intermediates reveals substrate‐directed reaction multiplicity
Author(s) -
Lin KuanHung,
Lyu SyueYi,
Yeh HsienWei,
Li YiShan,
Hsu NingShian,
Huang ChunMan,
Wang YungLin,
Shih HaoWei,
Wang ZheChong,
Wu ChangJer,
Li TsungLin
Publication year - 2020
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.3879
Subject(s) - flavin group , decarboxylation , chemistry , photochemistry , stereochemistry , reaction intermediate , zwitterion , monooxygenase , adduct , enzyme , molecule , catalysis , organic chemistry , cytochrome p450
Though reactive flavin‐N5/C4α‐oxide intermediates can be spectroscopically profiled for some flavin‐assisted enzymatic reactions, their exact chemical configurations are hardly visualized. Structural systems biology and stable isotopic labelling techniques were exploited to correct this stereotypical view. Three transition‐like complexes, the α‐ketoacid…N5‐FMN ox complex ( I ), the FMN ox ‐N5‐aloxyl‐C′α − ‐C4α + zwitterion ( II ), and the FMN‐N5‐ethenol‐N5‐C4α‐epoxide ( III ), were determined from mandelate oxidase (Hmo) or its mutant Y128F (monooxygenase) crystals soaked with monofluoropyruvate (a product mimic), establishing that N5 of FMN ox an alternative reaction center can polarize to an ylide‐like mesomer in the active site. In contrast, four distinct flavin‐C4α‐oxide adducts ( IV – VII ) from Y128F crystals soaked with selected substrates materialize C4α of FMN an intrinsic reaction center, witnessing oxidation, Baeyer–Villiger/peroxide‐assisted decarboxylation, and epoxidation reactions. In conjunction with stopped‐flow kinetics, the multifaceted flavin‐dependent reaction continuum is physically dissected at molecular level for the first time.