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Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate.
Holmes, Jacob B; Liu, Viktoriia; Caulkins, Bethany G; Hilario, Eduardo; Ghosh, Rittik K; Drago, Victoria N; Young, Robert P; Romero, Jennifer A; Gill, Adam D; Bogie, Paul M; Paulino, Joana; Wang, Xiaoling; Riviere, Gwladys; Bosken, Yuliana K; Struppe, Jochem; Hassan, Alia; Guidoulianov, Jevgeni; Perrone, Barbara; Mentink-Vigier, Frederic; Chang, Chia-En A; Long, Joanna R; Hooley, Richard J; Mueser, Timothy C; Dunn, Michael F; Mueller, Leonard J.
Afiliación
  • Holmes JB; Department of Chemistry, University of California, Riverside, CA 92521.
  • Liu V; Department of Chemistry, University of California, Riverside, CA 92521.
  • Caulkins BG; Department of Chemistry, University of California, Riverside, CA 92521.
  • Hilario E; W.M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges, Claremont, CA 91711.
  • Ghosh RK; Department of Chemistry, University of California, Riverside, CA 92521.
  • Drago VN; Department of Biochemistry, University of California, Riverside, CA 92521.
  • Young RP; Department of Chemistry and Biochemistry, University of Toledo, Toledo, OH 43606.
  • Romero JA; Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99354.
  • Gill AD; Department of Chemistry, University of California, Riverside, CA 92521.
  • Bogie PM; Department of Biochemistry, University of California, Riverside, CA 92521.
  • Paulino J; Department of Chemistry, University of California, Riverside, CA 92521.
  • Wang X; National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310.
  • Riviere G; National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310.
  • Bosken YK; Department of Biochemistry and Molecular Biology, McKnight Brain Institute, National High Magnetic Field Laboratory, University of Florida, Gainesville, FL 32610.
  • Struppe J; Department of Biochemistry, University of California, Riverside, CA 92521.
  • Hassan A; Bruker Biospin Corporation, Billerica, MA 01821.
  • Guidoulianov J; Bruker Switzerland AG 8117 Fällanden, Switzerland.
  • Perrone B; Bruker Switzerland AG 8117 Fällanden, Switzerland.
  • Mentink-Vigier F; Bruker Switzerland AG 8117 Fällanden, Switzerland.
  • Chang CA; National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310.
  • Long JR; Department of Chemistry, University of California, Riverside, CA 92521.
  • Hooley RJ; Department of Biochemistry and Molecular Biology, McKnight Brain Institute, National High Magnetic Field Laboratory, University of Florida, Gainesville, FL 32610.
  • Mueser TC; Department of Chemistry, University of California, Riverside, CA 92521.
  • Dunn MF; Department of Biochemistry, University of California, Riverside, CA 92521.
  • Mueller LJ; Department of Chemistry and Biochemistry, University of Toledo, Toledo, OH 43606.
Proc Natl Acad Sci U S A ; 119(2)2022 01 11.
Article en En | MEDLINE | ID: mdl-34996869
NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze ß-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate ß-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid-base catalytic residue ßLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cß and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Triptófano Sintasa / Espectroscopía de Resonancia Magnética / Cristalografía por Rayos X / Dominio Catalítico / Alanina Idioma: En Revista: Proc Natl Acad Sci U S A Año: 2022 Tipo del documento: Article Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Triptófano Sintasa / Espectroscopía de Resonancia Magnética / Cristalografía por Rayos X / Dominio Catalítico / Alanina Idioma: En Revista: Proc Natl Acad Sci U S A Año: 2022 Tipo del documento: Article Pais de publicación: Estados Unidos