title: Thiamin Diphosphate Activation in 1-Deoxy-d-xylulose 5-Phosphate Synthase: Insights into the Mechanism and Underlying Intermolecular Interactions.
abstract: 1-Deoxy-d-xylulose 5-phosphate synthase (DXS) is a thiamin diphosphate (TDP) dependent enzyme that marks the beginning of the methylerythritol 4-phosphate isoprenoid biosynthesis pathway. The mechanism of action for DXS is still poorly understood and begins with the formation of a thiazolium ylide. This TDP activation step is thought to proceed through an intramolecular deprotonation by the 4'-aminopyrimidine ring of TDP; however, this step would occur only after an initial deprotonation of its own 4'-amino group. The mechanism of the initial deprotonation has been hypothesized, by analogy to transketolases, to occur via a histidine or an active site water molecule. Results from hybrid quantum mechanical/molecular mechanical (QM/MM) reaction path calculations reveal an ∼10 kcal/mol difference in transition state energies, favoring a water mediated mechanism over direct deprotonation by histidine. This difference was determined to be largely governed by electrostatic changes induced by conformational variations in the active site. Additionally, mutagenesis studies reveal DXS to be an evolutionarily resilient enzyme. Particularly, we hypothesize that residues H82 and H304 may act in a compensatory fashion if the other is lost due to mutation. Further, nucleus-independent chemical shifts (NICSs) and aromatic stabilization energy (ASE) calculations suggest that reduction in TDP aromaticity also serves as a factor for regulating ylide formation and controlling reactivity.
authors.affiliation: Department of Chemistry, University of South Florida , 4202 E. Fowler Avenue, CHE205, Tampa, Florida 33620-5250, United States.
authors.forename: Justin K
authors.initials: JK
authors.lastname: White
authors.affiliation: Department of Chemistry, University of South Florida , 4202 E. Fowler Avenue, CHE205, Tampa, Florida 33620-5250, United States.|Department of Chemistry and Biochemistry, University of California, San Diego , 9500 Gilman Drive, La Jolla, California 92093-0332, United States.
authors.forename: Sumit
authors.initials: S
authors.lastname: Handa
authors.affiliation: Department of Chemistry, University of South Florida , 4202 E. Fowler Avenue, CHE205, Tampa, Florida 33620-5250, United States.
authors.forename: Sai Lakshmana
authors.initials: SL
authors.lastname: Vankayala
authors.affiliation: Department of Chemistry, University of South Florida , 4202 E. Fowler Avenue, CHE205, Tampa, Florida 33620-5250, United States.
authors.forename: David J
authors.initials: DJ
authors.lastname: Merkler
authors.affiliation: Department of Chemistry, University of South Florida , 4202 E. Fowler Avenue, CHE205, Tampa, Florida 33620-5250, United States.
authors.forename: H Lee
authors.initials: HL
authors.lastname: Woodcock
mesh_terms: D034301:Deinococcus / Q000201:enzymology; D011756:Diphosphates / Q000737:chemistry / Q000378:metabolism*; D008968:Molecular Conformation; D011789:Quantum Theory; D013831:Thiamine / Q000737:chemistry / Q000378:metabolism*; D014166:Transferases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011756:Diphosphates; D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase; D013831:Thiamine