Coq6 is an enzyme involved in the biosynthesis of coenzyme Q, a polyisoprenylated benzoquinone lipid essential to the function of the mitochondrial respiratory chain. In the yeast Saccharomyces cerevisiae, this putative flavin-dependent monooxygenase is proposed to hydroxylate the benzene ring of coenzyme Q (ubiquinone) precursor at position C5. We show here through biochemical studies that Coq6 is a flavoprotein using FAD as a cofactor. Homology models of the Coq6-FAD complex are constructed and studied through molecular dynamics and substrate docking calculations of 3-hexaprenyl-4-hydroxyphenol (4-HP6), a bulky hydrophobic model substrate. We identify a putative access channel for Coq6 in a wild type model and propose in silico mutations positioned at its entrance capable of partially (G248R and L382E single mutations) or completely (a G248R-L382E double-mutation) blocking access to the channel for the substrate. Further in vivo assays support the computational predictions, thus explaining the decreased activities or inactivation of the mutated enzymes. This work provides the first detailed structural information of an important and highly conserved enzyme of ubiquinone biosynthesis.
References
[1]
Bentinger M, Tekle M, Dallner G (2010) Coenzyme Q- biosynthesis and functions. Biochem Biophys Res Commun 396: 74–79. doi: 10.1016/j.bbrc.2010.02.147. pmid:20494114
[2]
Nowicka B, Kruk J (2010) Occurrence, biosynthesis and function of isoprenoid quinones. Biochim Biophys Acta 1797:1587–1605. doi: 10.1016/j.bbabio.2010.06.007. pmid:20599680
[3]
Aussel L, Pierrel F, Loiseau L, Lombard M, Fontecave M, Barras F (2014) Biosynthesis and physiology of coenzyme Q in bacteria. Biochim Biophys Acta, Bioenergetics 1837 (7): 1004–1011. doi: 10.1016/j.bbabio.2014.01.015. pmid:24480387
Kawamukai M (2009) Biosynthesis and bioproduction of coenzyme Q10 by yeasts and other organisms. Biotechnol Appl Biochem 53: 217–226. doi: 10.1042/BA20090035. pmid:19531029
[6]
Turunen M, Olsson J, Dallner G (2004) Metabolism and Function of coenzyme Q. Biochim Biophys Acta Biomembranes 1660: 171–199. Bentinger M, Brismar K, Dallner G (2007) The antioxidant role of coenzyme Q. Mitochondrion 7: S41–S50.
[7]
Quinzii CM, Hirano M (2010) Coenzyme Q and mitochondrial disease. Dev Disabil Res Rev 16:183–188. doi: 10.1002/ddrr.108. pmid:20818733
[8]
Hayashi K, Ogiyama Y, Yokomi K, Nakagawa T, Kaino T, Kawamukai M (2014) Functional conservation of coenzyme Q biosynthetic genes among yeasts, plants, and humans. PLoS One 9: e99038. doi: 10.1371/journal.pone.0099038. pmid:24911838
[9]
Meganathan R (2001) Ubiquinone Biosynthesis in Microorganisms. FEMS Microbiol Lett 203: 131–39. pmid:11583838 doi: 10.1111/j.1574-6968.2001.tb10831.x
[10]
Tran UC, Clarke CF (2007) Endogenous synthesis of coenzyme Q in eukaryotes. Mitochondrion 7: S62–71. pmid:17482885 doi: 10.1016/j.mito.2007.03.007
[11]
Wang Y, Hekimi S (2013) Molecular genetics of ubiquinone biosynthesis in animals. Crit Rev Biochem Mol Biol 48, 69–88. doi: 10.3109/10409238.2012.741564. pmid:23190198
[12]
Allan CM, Awad AM, Johnson JS, Shirasaki DI, Wang C, Blaby-Haas CE et al. (2015) Identification of Coq11, a new coenzyme Q biosynthetic protein in the CoQ-synthome in Saccharomyces cerevisiae. J Biol Chem 290: 7517–7534. doi: 10.1074/jbc.M114.633131. pmid:25631044
[13]
He CHW, Xie LTX, Allan CM, Tran UC, Clarke CF (2014) Coenzyme Q Supplementation or over-Expression of the Yeast Coq8 Putative Kinase Stabilizes Multi-Subunit Coq Polypeptide Complexes in Yeast Coq Null Mutants. Biochim Biophys Acta—Molecular and Cell Biology of Lipids 1841: 630–644. doi: 10.1016/j.bbalip.2013.12.017
[14]
Marbois B, Gin P, Gulmezian M, Clarke CF (2009) The yeast Coq4 polypeptide organizes a mitochondrial protein complex essential for the coenzyme Q biosynthesis, Biochim. Biophys. Acta—Molecular and Cell Biology of Lipids 1791, 69–75. doi: 10.1016/j.bbalip.2008.10.006
[15]
Gin P, Hsu AY, Rothman SC, Jonassen T, Lee PT, Tzagoloff A, Clarke CF (2003) The Saccharomyces Cerevisiae COQ6 Gene Encodes a Mitochondrial Flavin-Dependent Monooxygenase Required for Coenzyme Q Biosynthesis. J Biol Chem 278:25308–25316. pmid:12721307 doi: 10.1074/jbc.m303234200
[16]
Pierrel F, Hamelin O, Douki T, Kieffer-Jaquinod S, Mulhenhoff U, Ozeir M et al. (2010) Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis. Chem Biol 17: 449–459. doi: 10.1016/j.chembiol.2010.03.014. pmid:20534343
[17]
Ozeir M, Muhlenhoff U, Webert H, Lill R, Fontecave M, Pierrel F. (2011). Coenzyme Q Biosynthesis: Coq6 Is Required for the C5-Hydroxylation Reaction and Substrate Analogs Rescue Coq6 Deficiency. Chem Biol 18: 1134–1142. doi: 10.1016/j.chembiol.2011.07.008. pmid:21944752
[18]
Laredj LN, Licitra F, Puccio HM (2014) The molecular genetics of coenzyme Q biosynthesis in health and disease. Biochimie 100C: 78–87. doi: 10.1016/j.biochi.2013.12.006
[19]
Rotig A, Appelkvist EL, Geromel V, Chrétien D, Kadhom N, Edery P, et al. (2000) Quinone-responsive multiple respiratory-chain dysfunction due to widespread coenzyme Q10 deficiency. Lancet 356: 391–395. pmid:10972372 doi: 10.1016/s0140-6736(00)02531-9
[20]
Heeringa SF, Chernin G, Chaki M, Zhou W, Sloan AJ, Ji Z, et al. (2011) COQ6 mutations in human patients produce nephrotic syndrome with sensorineural deafness. J. Clin. Invest. 121: 2013–2024. doi: 10.1172/JCI45693. pmid:21540551
[21]
Consortium Uniprot (2015) UniProt: A Hub for Protein Information. Nucleic Acids Res 43: D204–12. doi: 10.1093/nar/gku989. pmid:25348405
[22]
Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10: 845–858. doi: 10.1038/nprot.2015.053. pmid:25950237
[23]
Kelley LA, Sternberg MJE (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4: 363–371. . doi: 10.1038/nprot.2009.2. pmid:19247286
[24]
Chehade MH, Loiseau L, Lombard M, Pecqueur L, Ismail A, Smadja M et al. (2013) UbiI, a new gene in Escherichia coli coenzyme Q biosynthesis, is involved in aerobic C5-hydroxylation. J. Biol.Chem. 288: 20085–20092. doi: 10.1074/jbc.M113.480368. pmid:23709220
[25]
Berman HM, Battistuz T, Bhat TN, Bluhm WF, Bourne PE, Buckhardt K et al. (2002). The Protein Data Bank. Acta Crystallogr D Biol Crystallogr 58: 899–907. pmid:12037327
[26]
Holm L, Rosenstr？m P (2010) Dali server: conservation mapping in 3D. Nucl Acids Res 38: W545–W549. doi: 10.1093/nar/gkq366. pmid:20457744
[27]
Oke M, Carter LG, Johnson KA, Liu H, McMahon SA, Yan X et al. (2010) The Scottish Structural Proteomics Facility: targets, methods and outputs. J. Struct Funct Genomics 11: 167–180. doi: 10.1007/s10969-010-9090-y. pmid:20419351
[28]
Accelrys Software Inc., Discovery Studio Modeling Environment, Release 4.0, San Diego: Accelrys Software Inc., 2013. .
[29]
Sievers F, Wilm A, Dineen DG, Gibson TJ, Karplus K, Li W et al. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539. . doi: 10.1038/msb.2011.75. pmid:21988835
[30]
Landau M, Mayrose I, Rosenberg Y, Glaser F, Martz E, Pupko T et al. (2005) ConSurf 2005: The projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Res 33, W299？W302. . pmid:15980475
[31]
Glaser F, Pupko T, Paz I, Bell RE, Bechor D, Martz E et al. (2003) ConSurf: Identification of functional regions in proteins by surface-mapping of phylogenetic information. Bioinformatics 19:163？164. pmid:12499312 doi: 10.1093/bioinformatics/19.1.163
[32]
？ali A, Blundell TL (1993). Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol, 234: 779–815. pmid:8254673
[33]
Fiser A, Do RKG, ？ali A (2000). Modeling of loops in protein structures. Protein Science 9: 1753–1773. pmid:11045621 doi: 10.1110/ps.9.9.1753
[34]
Eswar N, Marti-Renom MA, Webb B, Madhusudhan MS, Eramian D, Shen M, Pieper U, Sali A. Comparative Protein Structure Modeling With MODELLER. Curr Protoc Bioinformatics, John Wiley & Sons, Inc. Chapter 5: 5.6.1–5.6.30, 2006.
Roy A, Kucukural A, Zhang Y (2010) I-TASSER: a unified platform for automated Protein structure and function prediction. Nat Methods 5: 725–738. See also: Yang JY, Yan RX, Roy A, Xu D, Poisson J, Zhang Y (2015) The I-TASSER Suite: Protein structure and function prediction. Nat Methods 12: 7–8
[37]
Kim DE, Chivian D, Baker D (2004) Protein structure prediction and analysis using the Robetta server. Nucleic Acids Res 32: W526–W531. robetta.bakerlab.org. pmid:15215442 doi: 10.1093/nar/gkh468
[38]
Mullins JG (2012) Structural modelling pipelines in next generation sequencing projects. Adv Protein Chem Struct Biol. 89:117–167. doi: 10.1016/B978-0-12-394287-6.00005-7. pmid:23046884
[39]
Hornak V, Abel R, Okur A, Strockbine B, Roitberg A, Simmerling C (2006) Comparison of multiple amber force fields and development of improved protein backbone parameters. Proteins Struct Funct Bioinf 65:712. See also: Lindorff-Larse K, Piana S, Palmo K, Maragakis P, Klepeis JL, Dror RO et al. (2010) Improved Side-Chain Torsion Potentials for the Amber ff99SB Protein Force Field. Proteins 78: 1950–58. doi: 10.1002/prot.21123
[40]
Schreuder HA, Prick PAJ, Wierenga RK, Vriend G, Wilson KS, WGJ Hol et al. (1989) Crystal Structure of the P-Hydroxybenzoate Hydroxylase-Substrate Complex Refined at 1.9 ？ Resolution: Analysis of the Enzyme-Substrate and Enzyme-Product Complexes. J Mol Biol 208: 679–696. pmid:2553983 doi: 10.1016/0022-2836(89)90158-7
[41]
Sengupta A, Sasikala WD, Mukherjee A, Hazra P (2012) Comparative Study of Flavins Binding with Human Serum Albumin: A Fluorometric, Thermodynamic, and Molecular Dynamics Approach.” ChemPhysChem 13: 2142–2153. doi: 10.1002/cphc.201200044. pmid:22532419
[42]
Wang JM, Wolf RM, Caldwell JW, Kollman PA, Case DA (2004) Development and testing of a general amber force field. J Comput Chem 25: 1157–1174. pmid:15116359
[43]
Jorgensen WL, Jenson C (1998) Temperature dependence of TIP3P, SPC, and TIP4P water form NPT Monte Carlo simulations: seeking temperatures of maximal density. J Comp Chem 19: 1179–1186. doi: 10.1002/(sici)1096-987x(19980730)19:10<1179::aid-jcc6>3.0.co;2-j
[44]
Ballantyne JS, Moyes CD (1987) The Effects of Salinity Acclimation on the Osmotic Properties of Mitochondria from the Gill of Crassostrea-Virginica. J Exp Biol 133: 449–456.
[45]
Pronk S, Páll S, Schulz R, Larsson P, Bjelkmar P, Apostolov R et al. (2013) GROMACS 4.5: A High-Throughput and Highly Parallel Open Source Molecular Simulation Toolkit. Bioinformatics 29 (7): 845–54. doi: 10.1093/bioinformatics/btt055. pmid:23407358
[46]
Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJC (2005) GROMACS: Fast, Flexible, and Free. J Comput Chem 26: 1701–1718. pmid:16211538
[47]
Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG (1995) A Smooth Particle Mesh Ewald Method. J Chem Phys 103(19): 8577–8593. doi: 10.1063/1.470117
[48]
Páll S, Hess B (2013) A flexible algorithm for calculating pair interactions on SIMD architectures. Comput Phys Commun 184, 2641–2650. doi: 10.1016/j.cpc.2013.06.003
Parrinello M, Rahman A (1981) Polymorphic Transitions in Single Crystals: A New Molecular Dynamics Method. J Appl Phys 52 (12): 7182–90. doi: 10.1063/1.328693
[51]
Hess B, Bekker H, Berendsen HJC, Fraaije JGEM (1997) LINCS: A Linear Constraint Solver for Molecular Simulations. J Comput Chem 18(12): 1463–1472. doi: 10.1002/(sici)1096-987x(199709)18:12<1463::aid-jcc4>3.3.co;2-l
[52]
Hess B (2008) P-LINCS: A Parallel Linear Constraint Solver for Molecular Simulation. J Chem Theory Comput 4(1): 116–22. doi: 10.1021/ct700200b. pmid:26619985
[53]
Humphrey W, Dalke A, Schulten K (1996). VMD: visual molecular dynamics. J Mol Graph, 14(1), 33–38. pmid:8744570 doi: 10.1016/0263-7855(96)00018-5
[54]
Chovancova E, Pavelka A, Benes P, Strnad O, Brezovsky J, Kozlikova B et al. (2012) CAVER 3.0: A Tool for the Analysis of Transport Pathways in Dynamic Protein Structures. PloS Comput Biol 8(10): e1002708. . doi: 10.1371/journal.pcbi.1002708. pmid:23093919
[55]
The PyMOL Molecular Graphics System, Version 1.7.4 Schr？dinger, LLC. .
[56]
Entsch B, Cole LJ, Ballou DP (2005) Protein Dynamics and Electrostatics in the Function of P-Hydroxybenzoate Hydroxylase. Arch Biochem Biophys 433: 297–311. pmid:15581585 doi: 10.1016/j.abb.2004.09.029
Trott O, Olson AJ (2010) AutoDock Vina: Improving the Speed and Accuracy of Docking with a New Scoring Function, Efficient Optimization, and Multithreading. J Comput Chem 31: 455–461. . doi: 10.1002/jcc.21334. pmid:19499576
[59]
Doimo M, Trevisson E, Airik R, Bergdoll M, Santos-Ocana C, Hildebrandt F et al. (2014) Effect of vanillic acid on COQ6 mutants identified in patients with coenzyme Q deficiency. Biochim Biophys Acta 1842: 1–6. doi: 10.1016/j.bbadis.2013.10.007. pmid:24140869
[60]
Gin P, Hsu AY, Rothman SC, Jonassen T, Lee PT, Tzagoloff A et al. (2003) The Saccharomyces cerevisiae COQ6 gene encodes a mitochondrial flavin-dependent monooxygenase required for coenzyme Q biosynthesis. J Biol Chem 278: 25308–25316. pmid:12721307 doi: 10.1074/jbc.m303234200
[61]
Burke D, Dawson D, Stearns T. Methods in Yeast Genetics: a Cold Spring Harbor Laboratory course manual, CSHL Press, Plainview, NY; 2000.
[62]
Bradford MM (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry 72: 248–252. pmid:942051
[63]
Lomize AL, Pogozheva ID, Lomize MA, Mosberg HI (2009) Positioning of proteins in membranes: a computational approach. Protein Science 15, 1318–1333. doi: 10.1110/ps.062126106
Battey JND, Kopp J, Bordoli L, Read RJ, Clarke ND, Schwede T (2007) Automated Server Predictions in CASP7. Proteins: Structure, Function, and Bioinformatics 69, no. S8: 68–82. doi: 10.1002/prot.21761
[66]
In this case, the scoring function was defined as the sum of the differences between the 1PBE active site interatomic distances (from XRD) and those sampled from MD of the pHBH-FAD apo complex. A low score indicated a high structural similarity between the substrate-free PHBH conformations sampled during MD and the substrate-bound PHBH active site in 1PBE structure.
[67]
Clarke CF (2000) New advances in coenzyme Q biosynthesis. Protoplasma. 213:134–147. doi: 10.1007/bf01282151
[68]
Zhang KQ, Lin JW, Wang JH, Wu XW, Gao HL, Hsieh YC et al. (2014) A germline missense mutation in COQ6 is associated with susceptibility to familial schwannomatosis. Genet Med 16(10): 787–792. doi: 10.1038/gim.2014.39. pmid:24763291
[69]
van Berkel WJH, Kamerbeek NM, Fraaije MW (2006) Flavoprotein monooxygenases, a diverse class of oxidative biocatalysts. J Biotechnol. 124:670–89. pmid:16712999 doi: 10.1016/j.jbiotec.2006.03.044
Ballou DP, Entsch B, Cole LJ (2005) Dynamics Involved in Catalysis by Single-Component and Two-Component Flavin-Dependent Aromatic Hydroxylases. Biochem Biophys Res Comm 338: 590–98. pmid:16236251 doi: 10.1016/j.bbrc.2005.09.081
[72]
Boehr DD, Nussinov R, Wright PE (2009) The role of dynamics conformational ensembles in biomolecular recognition. Nat Chem Biol 5:789–796. doi: 10.1038/nchembio.232. pmid:19841628
[73]
Montersino S, Orru R, Barendregt A, Westphal AH, Van Duijn , Mattevi A, van Berkel WJH (2013) Crystal Structure of 3-Hydroxybenzoate 6-Hydroxylase Uncovers Lipid-assisted Flavoprotein Strategy for Regioselective Aromatic Hydroxylation. J Biol Chem 288: 26235–26245. doi: 10.1074/jbc.M113.479303. pmid:23864660
