title: Understanding the Catalytic Mechanism of Xanthosine Methyltransferase in Caffeine Biosynthesis from QM/MM Molecular Dynamics and Free Energy Simulations.
abstract: S-Adenosyl-l-methionine (SAM) dependent xanthosine methyltransferase (XMT) is the key enzyme that catalyzes the first methyl transfer in the caffeine biosynthesis pathway to produce the intermediate 7-methylxanthosine (7mXR). Although XMT has been a subject of extensive discussions, the catalytic mechanism and nature of the substrate involved in the catalysis are still unclear. In this paper, quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy (potential of mean force or PMF) simulations are undertaken to determine the catalytic mechanism of the XMT-catalyzed reaction. Both xanthosine and its monoanionic form with N3 deprotonated are used as the substrates for the methylation. It is found that while the methyl group can be transferred to the monoanionic form of xanthosine with a reasonable free energy barrier (about 17 kcal/mol), that is not the case for the neutral xanthosine. The results suggest that the substrate for the first methylation step in the caffeine biosynthesis pathway is likely to be the monoanionic form of xanthosine rather than the neutral form as widely adopted. This conclusion is supported by the pKa value on N3 of xanthosine both measured in aqueous phase and calculated in the enzymatic environment. The structural and dynamics information from both the X-ray structure and MD simulations is also consistent with the monoanionic xanthosine scenario. The implications of this conclusion for caffeine biosynthesis are discussed.
authors.affiliation: Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee , Knoxville, Tennessee 37996, United States.|UT/ORNL Center for Molecular Biophysics, Oak Ridge National Laboratory , Oak Ridge, Tennessee 37830, United States.|Chemistry and Material Science Faculty, Shandong Agricultural University , Tai'an 271018, Shandong, People's Republic of China.
authors.forename: Ping
authors.initials: P
authors.lastname: Qian
authors.affiliation: Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee , Knoxville, Tennessee 37996, United States.
authors.forename: Hao-Bo
authors.initials: HB
authors.lastname: Guo
authors.affiliation: Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee , Knoxville, Tennessee 37996, United States.|UT/ORNL Center for Molecular Biophysics, Oak Ridge National Laboratory , Oak Ridge, Tennessee 37830, United States.
authors.forename: Yufei
authors.initials: Y
authors.lastname: Yue
authors.affiliation: Chemistry and Material Science Faculty, Shandong Agricultural University , Tai'an 271018, Shandong, People's Republic of China.
authors.forename: Liang
authors.initials: L
authors.lastname: Wang
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory , Oak Ridge, Tennessee 37831, United States.
authors.forename: Xiaohan
authors.initials: X
authors.lastname: Yang
authors.affiliation: Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee , Knoxville, Tennessee 37996, United States.|UT/ORNL Center for Molecular Biophysics, Oak Ridge National Laboratory , Oak Ridge, Tennessee 37830, United States.
authors.forename: Hong
authors.initials: H
authors.lastname: Guo
mesh_terms: D055162:Biocatalysis; D002110:Caffeine / Q000096:biosynthesis*; D008780:Methyltransferases / Q000737:chemistry / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D011522:Protons; D011789:Quantum Theory; D012263:Ribonucleosides / Q000378:metabolism*; D013816:Thermodynamics; D014970:Xanthines
chemical_list: D011522:Protons; D012263:Ribonucleosides; D014970:Xanthines; D002110:Caffeine; C005893:xanthosine; D008780:Methyltransferases