title: X-ray and NMR crystallography in an enzyme active site: the indoline quinonoid intermediate in tryptophan synthase.
abstract: Chemical-level details such as protonation and hybridization state are critical for understanding enzyme mechanism and function. Even at high resolution, these details are difficult to determine by X-ray crystallography alone. The chemical shift in NMR spectroscopy, however, is an extremely sensitive probe of the chemical environment, making solid-state NMR spectroscopy and X-ray crystallography a powerful combination for defining chemically detailed three-dimensional structures. Here we adopted this combined approach to determine the chemically rich crystal structure of the indoline quinonoid intermediate in the pyridoxal-5'-phosphate-dependent enzyme tryptophan synthase under conditions of active catalysis. Models of the active site were developed using a synergistic approach in which the structure of this reactive substrate analogue was optimized using ab initio computational chemistry in the presence of side-chain residues fixed at their crystallographically determined coordinates. Various models of charge and protonation state for the substrate and nearby catalytic residues could be uniquely distinguished by their calculated effects on the chemical shifts measured at specifically (13)C- and (15)N-labeled positions on the substrate. Our model suggests the importance of an equilibrium between tautomeric forms of the substrate, with the protonation state of the major isomer directing the next catalytic step.
authors.affiliation: Department of Chemistry, University of California, Riverside, California 92521, USA.
authors.forename: Jinfeng
authors.initials: J
authors.lastname: Lai
authors.forename: Dimitri
authors.initials: D
authors.lastname: Niks
authors.forename: Yachong
authors.initials: Y
authors.lastname: Wang
authors.forename: Tatiana
authors.initials: T
authors.lastname: Domratcheva
authors.forename: Thomas R M
authors.initials: TR
authors.lastname: Barends
authors.forename: Friedrich
authors.initials: F
authors.lastname: Schwarz
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Olsen
authors.forename: Douglas W
authors.initials: DW
authors.lastname: Elliott
authors.forename: M Qaiser
authors.initials: MQ
authors.lastname: Fatmi
authors.forename: Chia-en A
authors.initials: CE
authors.lastname: Chang
authors.forename: Ilme
authors.initials: I
authors.lastname: Schlichting
authors.forename: Michael F
authors.initials: MF
authors.lastname: Dunn
authors.forename: Leonard J
authors.initials: LJ
authors.lastname: Mueller
mesh_terms: D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D007211:Indoles / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000378:metabolism*; D025101:Vitamin B 6 / Q000378:metabolism
chemical_list: D007211:Indoles; C057812:indoline; D025101:Vitamin B 6; D014367:Tryptophan Synthase