Mechanistic insights into the conversion of flavin adenine dinucleotide (FAD) to 8-formyl FAD in formate oxidase: a combined experimental and in-silico study.
8-Formyl flavin adenine dinucleotide
Formate oxidase
Molecular dynamics simulation
Oxidative maturation
QM/MM umbrella sampling simulation
Journal
Bioresources and bioprocessing
ISSN: 2197-4365
Titre abrégé: Bioresour Bioprocess
Pays: Germany
ID NLM: 101665551
Informations de publication
Date de publication:
10 Jul 2024
10 Jul 2024
Historique:
received:
02
05
2024
accepted:
02
07
2024
medline:
10
7
2024
pubmed:
10
7
2024
entrez:
10
7
2024
Statut:
epublish
Résumé
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
Identifiants
pubmed: 38985371
doi: 10.1186/s40643-024-00782-4
pii: 10.1186/s40643-024-00782-4
doi:
Types de publication
Journal Article
Langues
eng
Pagination
67Subventions
Organisme : National Natural Science Foundation of China
ID : 32201030
Organisme : National Natural Science Foundation of China
ID : 32271319
Organisme : National Natural Science Foundation of China
ID : 32071267
Organisme : Science and Technology Department of Jilin Province
ID : 20230402041GH
Organisme : Science and Technology Department of Jilin Province
ID : YDZJ202301ZYTS537
Organisme : Education Department of Jilin Province
ID : JJKH20220970KJ
Organisme : Development and Reform Commission of Jilin Province
ID : 2023C015
Organisme : Fundamental Research Funds of the Central Universities in China
ID : 2024-JCXK-11
Informations de copyright
© 2024. The Author(s).
Références
Augustin P, Toplak M, Fuchs K et al (2018) Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein. J Biol Chem 293(8):2829–2840
pubmed: 29301933
pmcid: 5827430
doi: 10.1074/jbc.RA117.000846
Berendsen HJC, Postma JPM, van Gunsteren WF et al (1984) Molecular dynamics with coupling to an external bath. J Chem Phys 81(8):3684–3690
doi: 10.1063/1.448118
Blay V, Pei D (2019) Serine proteases: how did chemists tease out their catalytic mechanism? Chem Texts 5(4):19
Darden T, York D, Pedersen L (1993) Particle mesh Ewald: an N⋅log(N) method for Ewald sums in large systems. J Chem Phys 98(12):10089–10092
doi: 10.1063/1.464397
De Vivo M, Dal Peraro M, Klein ML (2008) Phosphodiester cleavage in ribonuclease H occurs via an associative two-metal-aided catalytic mechanism. J Am Chem Soc 130(33):10955–10962
pubmed: 18662000
pmcid: 2745632
doi: 10.1021/ja8005786
Doubayashi D, Ootake T, Maeda Y et al (2011) Formate oxidase, an enzyme of the glucose-methanol-choline oxidoreductase family, has a his-arg pair and 8-formyl-FAD at the catalytic site. Biosci Biotech Bioch 75(9):1662–1667
doi: 10.1271/bbb.110153
Doubayashi D, Oki M, Mikami B et al (2019) The microenvironment surrounding FAD mediates its conversion to 8-formyl-FAD in aspergillus oryzae RIB40 formate oxidase. J Biochem 166(1):67–75
pubmed: 30715389
doi: 10.1093/jb/mvz009
Edmondson DE (1974) Intramolecular hemiacetal formation in 8-formylriboflavine. Biochemistry 13(14):2817–2821
pubmed: 4407611
doi: 10.1021/bi00711a006
Elstner M, Porezag D, Jungnickel G et al (1998) Self-consistent-charge density-functional tight-binding method for simulations of complex materials properties. Phys Rev B 58(11):7260–7268
doi: 10.1103/PhysRevB.58.7260
Götz AW, Williamson MJ, Xu D et al (2012) Routine microsecond molecular dynamics simulations with AMBER on GPUs. 1. Generalized born. J Chem Theory Comput 8(5):1542–1555
pubmed: 22582031
pmcid: 3348677
doi: 10.1021/ct200909j
Heath RS, Turner NJ (2022) Recent advances in oxidase biocatalysts: enzyme discovery, cascade reactions and scale up. Curr Opin Green Sustain Chem 38:100693
doi: 10.1016/j.cogsc.2022.100693
Heuts DPHM, Scrutton NS, McIntire WS et al (2009) What’s in a covalent bond? FEBS J 276(13):3405–3427
pubmed: 19438712
doi: 10.1111/j.1742-4658.2009.07053.x
Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14(1):33–38
pubmed: 8744570
doi: 10.1016/0263-7855(96)00018-5
Kästner J, Senn HM, Thiel S et al (2006) QM/MM free-energy perturbation compared to thermodynamic integration and umbrella sampling: application to an enzymatic reaction. J Chem Theory Comput 2(2):452–461
pubmed: 26626532
doi: 10.1021/ct050252w
Konjik V, Brünle S, Demmer U et al (2017) The crystal structure of RosB: insights into the reaction mechanism of the first member of a family of flavodoxin-like enzymes. Angew Chem Int Ed 56(4):1146–1151
doi: 10.1002/anie.201610292
Kräutler V, van Gunsteren WF, Hünenberger PH (2001) A fast SHAKE algorithm to solve distance constraint equations for small molecules in molecular dynamics simulations. J Comput Chem 22(5):501–508
doi: 10.1002/1096-987X(20010415)22:5<501::AID-JCC1021>3.0.CO;2-V
Kumar S, Rosenberg JM, Bouzida D et al (1992) THE weighted histogram analysis method for free-energy calculations on biomolecules. I. The method. J Comput Chem 13(8):1011–1021
doi: 10.1002/jcc.540130812
Lao YE, Heyerdahl F, Jacobsen D et al (2022) An enzymatic assay with formate oxidase for point-of-care diagnosis of methanol poisoning. Basic Clin Pharmacol 131(6):547–554
doi: 10.1111/bcpt.13789
Leys D, Scrutton NS (2016) Sweating the assets of flavin cofactors: new insight of chemical versatility from knowledge of structure and mechanism. Curr Opin Struc Biol 41:19–26
doi: 10.1016/j.sbi.2016.05.014
Li R, Zhang S, Li Q et al (2022) An atom-economic enzymatic cascade catalysis for high-throughput RAFT synthesis of ultrahigh molecular weight polymers. Angew Chem Int Ed 61(46):e202213396
doi: 10.1002/anie.202213396
Linke JA, Rayat A, Ward JM (2023) Production of indigo by recombinant bacteria. Bioresour Bioprocess 10(1):20
pubmed: 36936720
pmcid: 10011309
doi: 10.1186/s40643-023-00626-7
Maeda Y, Doubayashi D, Oki M et al (2009a) The presence of a modified FAD in formate oxidase from Debaryomyces Vanjiriae MH201 and aspergillus oryzae RIB40. J Biosci Bioeng 108:S106
doi: 10.1016/j.jbiosc.2009.08.310
Maeda Y, Doubayashi D, Oki M et al (2009b) Expression in Escherichia coli of an unnamed protein gene from aspergillus oryzae RIB40 and cofactor analyses of the gene product as formate oxidase. Biosci Biotech Bioch 73(12):2645–2649
doi: 10.1271/bbb.90497
Maeda Y, Doubayashi D, Ootake T et al (2010) Crystallization and preliminary X-ray analysis of formate oxidase, an enzyme of the glucose-methanol-choline oxidoreductase family. Acta Cryst F 66(9):1064–1066
doi: 10.1107/S1744309110028605
Marcos-Alcalde I, Setoain J, Mendieta-Moreno JI et al (2015) MEPSA: minimum energy pathway analysis for energy landscapes. Bioinformatics 31(23):3853–3855
pubmed: 26231428
doi: 10.1093/bioinformatics/btv453
Mark P, Nilsson L (2001) Structure and dynamics of the TIP3P, SPC, and SPC/E water models at 298 K. J Phys Chem A 105(43):9954–9960
doi: 10.1021/jp003020w
Niehaus TA, Elstner M, Frauenheim T et al (2001) Application of an approximate density-functional method to sulfur containing compounds. J Mol Struc-Theochem 541(1):185–194
doi: 10.1016/S0166-1280(00)00762-4
Özpınar GA, Peukert W, Clark T (2010) An improved generalized AMBER force field (GAFF) for urea. J Mol Model 16(9):1427–1440
pubmed: 20162312
doi: 10.1007/s00894-010-0650-7
Robbins JM, Souffrant MG, Hamelberg D et al (2017) Enzyme-mediated conversion of flavin adenine dinucleotide (FAD) to 8-Formyl FAD in formate oxidase results in a modified cofactor with enhanced catalytic properties. Biochemistry 56(29):3800–3807
pubmed: 28640638
doi: 10.1021/acs.biochem.7b00335
Robbins JM, Bommarius AS, Gadda G (2018a) Mechanistic studies of formate oxidase from Aspergillus oryzae: a novel member of the glucose-methanol-choline oxidoreductase enzyme superfamily that oxidizes carbon acids. Arch Biochem Biophys 643:24–31
pubmed: 29458006
doi: 10.1016/j.abb.2018.02.007
Robbins JM, Geng J, Barry BA et al (2018b) Photoirradiation generates an ultrastable 8-formyl FAD semiquinone radical with unusual properties in formate oxidase. Biochemistry 57(40):5818–5826
pubmed: 30226367
doi: 10.1021/acs.biochem.8b00571
Roe DR, Cheatham TE III (2013) PTRAJ and CPPTRAJ: Software for processing and analysis of molecular dynamics trajectory data. J Chem Theory Comput 9(7):3084–3095
pubmed: 26583988
doi: 10.1021/ct400341p
Rosta E, Nowotny M, Yang W et al (2011) Catalytic mechanism of RNA backbone cleavage by ribonuclease H from quantum mechanics/molecular mechanics simulations. J Am Chem Soc 133(23):8934–8941
pubmed: 21539371
pmcid: 3110985
doi: 10.1021/ja200173a
Scott AP, Radom L (1996) Harmonic vibrational frequencies: an evaluation of Hartree-Fock, Møller-Plesset, quadratic configuration interaction, density functional theory, and semiempirical scale factors. J Phys Chem 100(41):16502–16513
doi: 10.1021/jp960976r
Seabra GM, Walker RC, Elstner M et al (2007) Implementation of the SCC-DFTB method for hybrid QM/MM simulations within the Amber molecular dynamics package. J Phys Chem A 111(26):5655–5664
doi: 10.1021/jp070071l
Srinivasan B (2022) A guide to the Michaelis-Menten equation: steady state and beyond. FEBS J 289(20):6086–6098
pubmed: 34270860
doi: 10.1111/febs.16124
Sun BY, Sui HL, Liu ZW et al (2022) Structure-guided engineering of a flavin-containing monooxygenase for the efficient production of indirubin. Bioresour Bioprocess 9(1):70
pubmed: 38647553
pmcid: 10991670
doi: 10.1186/s40643-022-00559-7
Tao Y, Zhao Q, Liu F et al (2024) Enzymes encapsulated in organic-inorganic hybrid nanoflower with spatial localization for sensitive and colorimetric detection of formate. J Colloid Interface Sci 672:97–106
pubmed: 38833738
doi: 10.1016/j.jcis.2024.05.231
Tian C, Kasavajhala K, Belfon KAA et al (2020) ff19SB: amino-acid-specific protein backbone parameters trained against quantum mechanics energy surfaces in solution. J Chem Theory Comput 16(1):528–552
pubmed: 31714766
doi: 10.1021/acs.jctc.9b00591
Willot SJP, Hoang MD, Paul CE et al (2020) FOx news: towards methanol-driven biocatalytic oxyfunctionalisation reactions. ChemCatChem 12(10):2713–2716
doi: 10.1002/cctc.202000197
Wongnate T, Chaiyen P (2013) The substrate oxidation mechanism of pyranose 2-oxidase and other related enzymes in the glucose-methanol-choline superfamily. FEBS J 280(13):3009–3027
pubmed: 23578136
doi: 10.1111/febs.12280
Yorita K, Matsuoka T, Misaki H et al (2000) Interaction of two arginine residues in lactate oxidase with the enzyme flavin: Conversion of FMN to 8-formyl-FMN. Proc Natl Acad Sci 97(24):13039–13044
pubmed: 11078532
pmcid: 27174
doi: 10.1073/pnas.250472297