title: Cryo-crystallography of a true substrate, indole-3-glycerol phosphate, bound to a mutant (alphaD60N) tryptophan synthase alpha2beta2 complex reveals the correct orientation of active site alphaGlu49.
abstract: The reversible cleavage of indole-3-glycerol by the alpha-subunit of tryptophan synthase has been proposed to be catalyzed by alphaGlu49 and alphaAsp60. Although previous x-ray crystallographic structures of the tryptophan synthase alpha2beta2 complex showed an interaction between the carboxylate of alphaAsp60 and the bound inhibitor indole-3-propanol phosphate, the carboxylate of alphaGlu49 was too distant to play its proposed role. To clarify the structural and functional roles of alphaGlu49, we have determined crystal structures of a mutant (alphaD60N) alpha2beta2 complex in the presence and absence of the true substrate, indole-3-glycerol phosphate. The enzyme in the crystal cleaves indole-3-glycerol phosphate very slowly at room temperature but not under cryo-conditions of 95 K. The structure of the complex with the true substrate obtained by cryo-crystallography reveals that indole-3-glycerol phosphate and indole-3-propanol phosphate have similar binding modes but different torsion angles. Most importantly, the side chain of alphaGlu49 interacts with 3-hydroxyl group of indole-3-glycerol phosphate as proposed. The movement of the side chain of alphaGlu49 into an extended conformation upon binding the true substrate provides evidence for an induced fit mechanism. Our results demonstrate how cryo-crystallography and mutagenesis can provide insight into enzyme mechanism.
authors.affiliation: Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: S
authors.initials: S
authors.lastname: Rhee
authors.forename: E W
authors.initials: EW
authors.lastname: Miles
authors.forename: D R
authors.initials: DR
authors.lastname: Davies
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D005994:Glycerophosphates / Q000737:chemistry / Q000378:metabolism*; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D005994:Glycerophosphates; D046911:Macromolecular Substances; D011994:Recombinant Proteins; C008117:indoleglycerol phosphate; D014367:Tryptophan Synthase