Mechanistic insights into the conversion of flavin adenine dinucleotide(FAD)to 8-formyl FAD in formate oxidase:a combined experimental and in-silico study  

作　　者：Kai Wen Sirui Wang Yixin Sun Mengsong Wang Yingjiu Zhang Jingxuan Zhu Quanshun Li 

机构地区：[1]Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education,School of Life Sciences,Jilin University,Changchun 130012,China [2]Center for Supramolecular Chemical Biology,Jilin University,Changchun 130012,China

出　　处：《Bioresources and Bioprocessing》2024年第1期909-921,共13页生物资源与生物加工(英文)

基　　金：supported by the National Natural Science Foundation of China(32201030,32271319 and 32071267);the Science and Technology Department of Jilin Province(20230402041GH and YDZJ202301ZYTS537);the Education Department of Jilin Province(JJKH20220970KJ);the Development and Reform Commission of Jilin Province(2023C015);the Fundamental Research Funds of the Central Universities in China(2024-JCXK-11).

摘　　要：Formate oxidase(FOx),which contains 8-formyl flavin adenine dinucleotide(FAD),exhibits a distinct advantage in utilizing ambient oxygen molecules for the oxidation of formic acid compared to other glucose-methanol-choline(GMC)oxidoreductase enzymes that contain only the standard FAD cofactor.The FOx-mediated conversion of FAD to 8-formyl FAD results in an approximate 10-fold increase in formate oxidase activity.However,the mechanistic details underlying the autocatalytic formation of 8-formyl FAD are still not well understood,which impedes further utilization of FOx.In this study,we employ molecular dynamics simulation,QM/MM umbrella sampling simulation,enzyme activity assay,site-directed mutagenesis,and spectroscopic analysis to elucidate the oxidation mechanism of FAD to 8-formyl FAD.Our results reveal that a catalytic water molecule,rather than any catalytic amino acids,serves as a general base to deprotonate the C8 methyl group on FAD,thus facilitating the formation of a quinone-methide tautomer intermediate.An oxygen molecule subsequently oxidizes this intermediate,resulting in a C8 methyl hydroperoxide anion that is protonated and dissociated to form OHC-RP and OH−.During the oxidation of FAD to 8-formyl FAD,the energy barrier for the rate-limiting step is calculated to be 22.8 kcal/mol,which corresponds to the required 14-hour transformation time observed experimentally.Further,the elucidated oxidation mechanism reveals that the autocatalytic formation of 8-formyl FAD depends on the proximal arginine and serine residues,R87 and S94,respectively.Enzymatic activity assay validates that the mutation of R87 to lysine reduces the kcat value to 75%of the wild-type,while the mutation to histidine results in a complete loss of activity.Similarly,the mutant S94I also leads to the deactivation of enzyme.This dependency arises because the nucleophilic OH−group and the quinone-methide tautomer intermediate are stabilized through the noncovalent interaction provided by R87 and S94.These findings not only explain th

关 键 词：Formate oxidase 8-Formyl flavin adenine dinucleotide Oxidative maturation Molecular dynamics simulation QM/MM umbrella sampling simulation 

分 类 号：O62[理学—有机化学]