The Pden_5119 protein oxidizes NADH with oxygen under mediation by the bound flavin mononucleotide (FMN) and may be involved in the maintenance of the cellular redox pool. In biochemical characterization, the curve of the pH-rate dependence was bell-shaped with pKa1 = 6.6 and pKa2 = 9.2 at 2 μM FMN while it contained only a descending limb pKa of 9.7 at 50 μM FMN. The enzyme was found to undergo inactivation by reagents reactive with histidine, lysine, tyrosine, and arginine. In the first three cases, FMN exerted a protective effect against the inactivation. X-ray structural analysis coupled with site-directed mutagenesis identified three amino acid residues important to the catalysis. Structural and kinetic data suggest that His-117 plays a role in the binding and positioning of the isoalloxazine ring of FMN, Lys-82 fixes the nicotinamide ring of NADH to support the proS-hydride transfer, and Arg-116 with its positive charge promotes the reaction between dioxygen and reduced flavin.

Department of Biochemistry Faculty of Science Masaryk University Kotlářská 2 61137 Brno Czech Republic

Zobrazit více v PubMed

Ross D., Siegel D. The diverse functionality of NQO1 and its roles in redox control. Redox Biol. 2021;41:101950. doi: 10.1016/j.redox.2021.101950. PubMed DOI PMC

Mazoch J., Tesarik R., Sedlacek V., Kucera I., Turanek J. Isolation and biochemical characterization of two soluble iron(III) reductases from Paracoccus denitrificans. Eur. J. Biochem. 2004;271:553–562. doi: 10.1046/j.1432-1033.2003.03957.x. PubMed DOI

Sedlacek V., van Spanning R.J.M., Kucera I. Characterization of the quinone reductase activity of the ferric reductase B protein from Paracoccus denitrificans. Arch. Biochem. Biophys. 2009;483:29–36. doi: 10.1016/j.abb.2008.12.016. PubMed DOI

Sedlacek V., Ptackova N., Rejmontova P., Kucera I. The flavoprotein FerB of Paracoccus denitrificans binds to membranes, reduces ubiquinone and superoxide, and acts as an in vivo antioxidant. FEBS J. 2015;282:283–296. doi: 10.1111/febs.13126. PubMed DOI

Sedlacek V., Kucera I. Arginine-95 is important for recruiting superoxide to the active site of the FerB flavoenzyme of Paracoccus denitrificans. FEBS Lett. 2019;593:697–702. doi: 10.1002/1873-3468.13359. PubMed DOI

Sedlacek V., Klumpler T., Marek J., Kucera I. The structural and functional basis of catalysis mediated by NAD(P)H:acceptor oxidoreductase (FerB) of Paracoccus denitrificans. PLoS ONE. 2014;9:e96262. doi: 10.1371/journal.pone.0096262. PubMed DOI PMC

Pernikarova V., Sedlacek V., Potesil D., Prochazkova I., Zdrahal Z., Bouchal P., Kucera I. Proteomic responses to a methyl viologen-induced oxidative stress in the wild type and FerB mutant strains of Paracoccus denitrificans. J. Proteom. 2015;125:68–75. doi: 10.1016/j.jprot.2015.05.002. PubMed DOI

Sedlacek V., Kucera I. Functional and mechanistic characterization of an atypical flavin reductase encoded by the pden_5119 gene in Paracoccus denitrificans. Mol. Microbiol. 2019;112:166–183. doi: 10.1111/mmi.14260. PubMed DOI

Sedlacek V., Kryl M., Kucera I. The ArsH protein product of the Paracoccus denitrificans ars operon has an activity of organoarsenic reductase and is regulated by a redox-responsive repressor. Antioxidants. 2022;11:902. doi: 10.3390/antiox11050902. PubMed DOI PMC

Edgcomb S.P., Murphy K.P. Variability in the pKa of histidine side-chains correlates with burial within proteins. Proteins. 2002;49:1–6. doi: 10.1002/prot.10177. PubMed DOI

Miles E.W. Modification of histidyl residues in proteins by diethylpyrocarbonate. Methods Enzymol. 1977;47:431–442. PubMed

Chen S.S., Engel P.C. Equilibrium position of reaction of bovine liver glutamate dehydrogenase with pyridoxal 5′-phosphate. Demonstration that covalent modification with this reagent completely abolishes catalytic activity. Biochem. J. 1975;147:351–358. doi: 10.1042/bj1470351. PubMed DOI PMC

Simpson R.T., Riordan J.F., Vallee B.L. Functional tyrosyl residues in active center of bovine pancreatic carboxypeptidase A. Biochemistry. 1963;2:616–622. doi: 10.1021/bi00903a039. PubMed DOI

Takahashi K. Reaction of phenylglyoxal with arginine residues in proteins. J. Biol. Chem. 1968;243:6171–6179. doi: 10.1016/S0021-9258(18)94475-3. PubMed DOI

Nissen M.S., Youn B.Y., Knowles B.D., Ballinger J.W., Jun S.Y., Belchik S.M., Xun L.Y., Kang C.H. Crystal structures of NADH:FMN oxidoreductase (EmoB) at different stages of catalysis. J. Biol. Chem. 2008;283:28710–28720. doi: 10.1074/jbc.M804535200. PubMed DOI PMC

Driggers C.M., Dayal P.V., Ellis H.R., Karplus P.A. Crystal structure of Escherichia coli SsuE: Defining a general catalytic cycle for FMN reductases of the flavodoxin-like superfamily. Biochemistry. 2014;53:3509–3519. doi: 10.1021/bi500314f. PubMed DOI

Pettersen E.F., Goddard T.D., Huang C.C., Couch G.S., Greenblatt D.M., Meng E.C., Ferrin T.E. UCSF chimera–A visualization system for exploratory research and analysis. J. Comput. Chem. 2004;25:1605–1612. doi: 10.1002/jcc.20084. PubMed DOI

Ellis H.R. The FMN-dependent two-component monooxygenase systems. Arch. Biochem. Biophys. 2010;497:1–12. doi: 10.1016/j.abb.2010.02.007. PubMed DOI

Robbins J.M., Ellis H.R. Investigations of two-component flavin-dependent monooxygenase systems. Methods Enzymol. 2019;620:399–422. PubMed

Li G.F., Glusac K.D. Light-triggered proton and electron transfer in flavin cofactors. J. Phys. Chem. A. 2008;112:4573–4583. doi: 10.1021/jp7117218. PubMed DOI

Rippa M., Spanio L., Pontremo S. A specific interaction of pyridoxal 5′-phosphate and 6-phosphogluconic dehydrogenase. Arch. Biochem. Biophys. 1967;118:48–57. doi: 10.1016/0003-9861(67)90276-7. DOI

Gould K.G., Engel P.C. Modification of mouse testicular lactate dehydrogenase by pyridoxal 5′-phosphate. Biochem. J. 1980;191:365–371. doi: 10.1042/bj1910365. PubMed DOI PMC

Lilley K.S., Engel P.C. The essential active-site lysines of clostridial glutamate dehydrogenase. A study with pyridoxal-5′-phosphate. Eur. J. Biochem. 1992;207:533–540. doi: 10.1111/j.1432-1033.1992.tb17079.x. PubMed DOI

Olano J., Soler J., Busto F., de Arriaga D. Chemical modification of NADP-isocitrate dehydrogenase from Cephalosporium acremonium. Evidence of essential histidine and lysine groups at the active site. Eur. J. Biochem. 1999;261:640–649. doi: 10.1046/j.1432-1327.1999.00297.x. PubMed DOI

Vazquez M.A., Munoz F., Donoso J., Garcia Blanco F. Stability of Schiff bases of amino acids and pyridoxal-5′-phosphate. Amino Acids. 1992;3:81–94. doi: 10.1007/BF00806010. PubMed DOI

Gillet N., Levy B., Moliner V., Demachy I., de la Lande A. Electron and hydrogen atom transfers in the hydride carrier protein EmoB. J. Chem. Theory Comput. 2014;10:5036–5046. doi: 10.1021/ct500173y. PubMed DOI

Romero E., Gomez Castellanos J.R., Gadda G., Fraaije M.W., Mattevi A. Same substrate, many reactions: Oxygen activation in flavoenzymes. Chem. Rev. 2018;118:1742–1769. doi: 10.1021/acs.chemrev.7b00650. PubMed DOI

Pandeswari P.B., Sabareesh V., Vijayalakshmi M.A. Insights into stoichiometry of arginine modification by phenylglyoxal and 1,2-cyclohexanedione probed by LC-ESI-MS. J. Prot. Proteom. 2016;7:323–347.

Cornish-Bowden A. Fundamentals of Enzyme Kinetics. 4th ed. Wiley-Blackwell; Hoboken, NJ, USA: 2012. p. 263.

Hu W., Xie J., Chau H.W. Evaluation of parameter uncertainties in nonlinear regression using Microsoft Excel Spreadsheet. Environ. Syst. Res. 2015;4:4. doi: 10.1186/s40068-015-0031-4. DOI

Melchior W.B., Fahrney D. Ethoxyformylation of proteins. Reaction of ethoxyformic anhydride with alpha-chymotrypsin, pepsin, and pancreatic ribonuclease at pH 4. Biochemistry. 1970;9:251–258. doi: 10.1021/bi00804a010. PubMed DOI

Kitz R., Wilson I.B. Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesterase. J. Biol. Chem. 1962;237:3245–3249. doi: 10.1016/S0021-9258(18)50153-8. PubMed DOI

Gomi T., Fujioka M. Evidence for an essential histidine residue in S-adenosylhomocysteinase from rat liver. Biochemistry. 1983;22:137–143. doi: 10.1021/bi00270a020. PubMed DOI

Lostao A., El Harrous M., Daoudi F., Romero A., Parody-Morreale A., Sancho J. Dissecting the energetics of the apoflavodoxin-FMN complex. J. Biol. Chem. 2000;275:9518–9526. doi: 10.1074/jbc.275.13.9518. PubMed DOI

Kabsch W. XDS. Acta Crystallogr. D Biol. Crystallogr. 2010;66:125–132. doi: 10.1107/S0907444909047337. PubMed DOI PMC

Winn M.D., Ballard C.C., Cowtan K.D., Dodson E.J., Emsley P., Evans P.R., Keegan R.M., Krissinel E.B., Leslie A.G., McCoy A., et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 2011;67:235–242. doi: 10.1107/S0907444910045749. PubMed DOI PMC

Murshudov G.N., Skubak P., Lebedev A.A., Pannu N.S., Steiner R.A., Nicholls R.A., Winn M.D., Long F., Vagin A.A. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 2011;67:355–367. doi: 10.1107/S0907444911001314. PubMed DOI PMC

Kovalevskiy O., Nicholls R.A., Murshudov G.N. Automated refinement of macromolecular structures at low resolution using prior information. Acta Crystallogr. D Struct. Biol. 2016;72:1149–1161. doi: 10.1107/S2059798316014534. PubMed DOI PMC

Joosten R.P., Salzemann J., Bloch V., Stockinger H., Berglund A.C., Blanchet C., Bongcam-Rudloff E., Combet C., Da Costa A.L., Deleage G., et al. PDB_REDO: Automated re-refinement of X-ray structure models in the PDB. J. Appl. Crystallogr. 2009;42:376–384. doi: 10.1107/S0021889809008784. PubMed DOI PMC

Afonine P.V., Poon B.K., Read R.J., Sobolev O.V., Terwilliger T.C., Urzhumtsev A., Adams P.D. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D Struct. Biol. 2018;74:531–544. doi: 10.1107/S2059798318006551. PubMed DOI PMC

Konarev P.V., Volkov V.V., Sokolova A.V., Koch M.H.J., Svergun D.I. PRIMUS: A Windows PC-based system for small-angle scattering data analysis. J. Appl. Crystallogr. 2003;36:1277–1282. doi: 10.1107/S0021889803012779. DOI

Svergun D., Barberato C., Koch M.H.J. CRYSOL–a program to evaluate x-ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Crystallogr. 1995;28:768–773. doi: 10.1107/S0021889895007047. DOI

Biasini M., Bienert S., Waterhouse A., Arnold K., Studer G., Schmidt T., Kiefer F., Gallo Cassarino T., Bertoni M., Bordoli L., et al. SWISS-MODEL: Modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 2014;42:W252–W258. doi: 10.1093/nar/gku340. PubMed DOI PMC

Svergun D.I. Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys. J. 1999;76:2879–2886. doi: 10.1016/S0006-3495(99)77443-6. PubMed DOI PMC

Emsley P., Cowtan K. Coot: Model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 2004;60:2126–2132. doi: 10.1107/S0907444904019158. PubMed DOI

Liu N., Xu Z. Using LeDock as a docking tool for computational drug design. IOP Conf. Ser. Earth Environ. Sci. 2018;218:012143. doi: 10.1088/1755-1315/218/1/012143. DOI