title: Quantum mechanics/molecular mechanics investigation of the chemical reaction in Dpo4 reveals water-dependent pathways and requirements for active site reorganization.
abstract: The nucleotidyl-transfer reaction coupled with the conformational transitions in DNA polymerases is critical for maintaining the fidelity and efficiency of DNA synthesis. We examine here the possible reaction pathways of a Y-family DNA polymerase, Sulfolobus solfataricus DNA polymerase IV (Dpo4), for the correct insertion of dCTP opposite 8-oxoguanine using the quantum mechanics/molecular mechanics (QM/MM) approach, both from a chemistry-competent state and a crystal closed state. The latter examination is important for understanding pre-chemistry barriers to interpret the entire enzyme mechanism, since the crystal closed state is not an ideal state for initiating the chemical reaction. The most favorable reaction path involves initial deprotonation of O3'H via two bridging water molecules to O1A, overcoming an overall potential energy barrier of approximately 20.0 kcal/mol. The proton on O1A-P(alpha) then migrates to the gamma-phosphate oxygen of the incoming nucleotide as O3' attacks P(alpha), and the P(alpha)-O3A bond breaks. The other possible pathway in which the O3'H proton is transferred directly to O1A on P(alpha) has an overall energy barrier of 25.0 kcal/mol. In both reaction paths, the rate-limiting step is the initial deprotonation, and the trigonal-bipyramidal configuration for P(alpha) occurs during the concerted bond formation (O3'-P(alpha)) and breaking (P(alpha)-O3A), indicating the associative nature of the chemical reaction. In contrast, the Dpo4/DNA complex with an imperfect active-site geometry corresponding to the crystal state must overcome a much higher activation energy barrier (29.0 kcal/mol) to achieve a tightly organized site due to hindered O3'H deprotonation stemming from larger distances and distorted conformation of the proton acceptors. This significant difference demonstrates that the pre-chemistry reorganization in Dpo4 costs approximately 4.0 to 9.0 kcal/mol depending on the primer terminus environment. Compared to the higher fidelity DNA polymerase beta from the X-family, Dpo4 has a higher chemical reaction barrier (20.0 vs 15.0 kcal/mol) due to the more solvent-exposed active site.
authors.affiliation: Department of Chemistry and Courant Institute of Mathematical Sciences, New York University, 251 Mercer Street, New York, New York 10012, USA.
authors.forename: Yanli
authors.initials: Y
authors.lastname: Wang
authors.forename: Tamar
authors.initials: T
authors.lastname: Schlick
mesh_terms: D055162:Biocatalysis; D020134:Catalytic Domain; D003198:Computer Simulation; D003460:Crystallization; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D010100:Oxygen / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011522:Protons; D011789:Quantum Theory; D048229:Sulfolobus solfataricus; D014867:Water / Q000737:chemistry*
chemical_list: D010710:Phosphates; D011522:Protons; D014867:Water; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D010100:Oxygen
references.citation: J Comput Chem. 2001 Nov 30;22(15):1843-1850
references.pmid: 12116415
references.citation: J Biol Chem. 2004 Aug 27;279(35):36951-61
references.pmid: 15210693
references.citation: Nature. 1998 Jan 15;391(6664):251-8
references.pmid: 9440688
references.citation: Biochemistry. 2007 Jan 16;46(2):461-71
references.pmid: 17209556
references.citation: Proc Natl Acad Sci U S A. 2005 May 10;102(19):6819-24
references.pmid: 15863620
references.citation: Biochemistry. 2006 Jun 13;45(23):7036-48
references.pmid: 16752894
references.citation: Biochemistry. 2005 Oct 11;44(40):13328-41
references.pmid: 16201758
references.citation: Annu Rev Biophys Biomol Struct. 2003;32:425-43
references.pmid: 12574064
references.citation: J Mol Biol. 2002 Apr 12;317(5):651-71
references.pmid: 11955015
references.citation: Biophys J. 1997 May;72(5):2075-93
references.pmid: 9129810
references.citation: Biophys J. 2004 Nov;87(5):3088-99
references.pmid: 15507687
references.citation: Nature. 2003 Aug 28;424(6952):1083-7
references.pmid: 12904819
references.citation: J Biol Chem. 2006 Jan 27;281(4):2358-72
references.pmid: 16306039
references.citation: Protein Sci. 2006 Jan;15(1):135-51
references.pmid: 16322565
references.citation: Chem Soc Rev. 2005 Oct;34(10):875-900
references.pmid: 16172677
references.citation: J Am Chem Soc. 2003 Jul 9;125(27):8163-77
references.pmid: 12837086
references.citation: Proc Natl Acad Sci U S A. 2007 Mar 13;104(11):4267-72
references.pmid: 17360513
references.citation: PLoS Biol. 2006 Jan;4(1):e11
references.pmid: 16379496
references.citation: Proteins. 2003 Nov 15;53(3):667-82
references.pmid: 14579358
references.citation: Proteins. 1986 Sep;1(1):47-59
references.pmid: 3449851
references.citation: Biochem Biophys Res Commun. 2006 Nov 24;350(3):521-9
references.pmid: 17022941
references.citation: Proteins. 2008 Jan 1;70(1):231-47
references.pmid: 17671961
references.citation: Phys Rev B Condens Matter. 1988 Jan 15;37(2):785-789
references.pmid: 9944570
references.citation: J Biol Chem. 2007 Dec 14;282(50):36421-33
references.pmid: 17951245
references.citation: Proc Natl Acad Sci U S A. 2008 Apr 15;105(15):5670-4
references.pmid: 18391201
references.citation: Biochemistry. 2004 Feb 24;43(7):2116-25
references.pmid: 14967051
references.citation: Cell. 2001 Oct 5;107(1):91-102
references.pmid: 11595188
references.citation: J Am Chem Soc. 2007 Sep 12;129(36):11100-10
references.pmid: 17696533
references.citation: Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):5970-5
references.pmid: 15069184
references.citation: Proc Natl Acad Sci U S A. 2006 Sep 5;103(36):13294-9
references.pmid: 16938895
references.citation: J Biol Chem. 1999 Jun 18;274(25):17395-8
references.pmid: 10364165
references.citation: J Biol Chem. 2005 Aug 19;280(33):29750-64
references.pmid: 15965231
references.citation: J Am Chem Soc. 2007 Apr 18;129(15):4731-7
references.pmid: 17375926
references.citation: Annu Rev Phys Chem. 2002;53:467-505
references.pmid: 11972016
references.citation: J Biol Chem. 2007 Jan 12;282(2):1456-67
references.pmid: 17105728
references.citation: Nature. 2000 Nov 23;408(6811):433-9
references.pmid: 11100718
references.citation: Science. 1994 Dec 23;266(5193):2022-5
references.pmid: 7528445
references.citation: Nucleic Acids Res. 2001 Nov 15;29(22):4607-16
references.pmid: 11713310
references.citation: Mol Cell. 2001 Jul;8(1):7-8
references.pmid: 11515498
references.citation: J Phys Chem B. 2008 Jan 24;112(3):1007-15
references.pmid: 18166038
references.citation: Biochemistry. 1997 Sep 16;36(37):11205-15
references.pmid: 9287163
references.citation: J Phys Chem B. 2005 Aug 18;109(32):15645-50
references.pmid: 16852982
references.citation: Chem Res Toxicol. 2005 Mar;18(3):441-56
references.pmid: 15777084
references.citation: Biochemistry. 2006 Dec 26;45(51):15142-56
references.pmid: 17176036
references.citation: EMBO J. 2005 Sep 7;24(17):2957-67
references.pmid: 16107880
references.citation: J Biol Chem. 2007 Jul 6;282(27):19831-43
references.pmid: 17468100
references.citation: Structure. 2006 Apr;14(4):757-66
references.pmid: 16615916
references.citation: Proc Natl Acad Sci U S A. 2004 Feb 24;101(8):2265-9
references.pmid: 14982998
references.citation: Mol Cell. 2004 Mar 12;13(5):751-62
references.pmid: 15023344
references.citation: Biophys J. 2002 Oct;83(4):1731-48
references.pmid: 12324397
references.citation: J Phys Chem B. 2007 Sep 27;111(38):11244-52
references.pmid: 17764165
references.citation: Nature. 2003 Jan 23;421(6921):436-40
references.pmid: 12540918