Please use this identifier to cite or link to this item: https://hdl.handle.net/11147/5650
Title: 1.55 Å-resolution structure of ent-copalyl diphosphate synthase and exploration of general acid function by site-directed mutagenesis
Authors: Köksal, Mustafa
Christianson, David W.
Peters, Reuben John
Potter, Kevin
Keywords: Enzyme mechanism
Gibberellin biosynthesis
Protein crystallography
Terpene cyclase
Issue Date: 2014
Publisher: Elsevier Ltd.
Source: Köksal, M., Potter, K., Peters, R.J., and Christianson, D.W. (2014). 1.55 Å-resolution structure of ent-copalyl diphosphate synthase and exploration of general acid function by site-directed mutagenesis. Biochimica et Biophysica Acta - General Subjects,1840(1), 184-190. doi:10.1016/j.bbagen.2013.09.004
Abstract: Background The diterpene cyclase ent-copalyl diphosphate synthase (CPS) catalyzes the first committed step in the biosynthesis of gibberellins. The previously reported 2.25 Å resolution crystal structure of CPS complexed with (S)-15-aza-14,15-dihydrogeranylgeranyl thiolodiphosphate (1) established the αβγ domain architecture, but ambiguities regarding substrate analog binding remained. Method Use of crystallization additives yielded CPS crystals diffracting to 1.55 Å resolution. Additionally, active site residues that hydrogen bond with D379, either directly or through hydrogen bonded water molecules, were probed by mutagenesis. Results This work clarifies structure-function relationships that were ambiguous in the lower resolution structure. Well-defined positions for the diphosphate group and tertiary ammonium cation of 1, as well as extensive solvent structure, are observed. Conclusions Two channels involving hydrogen bonded solvent and protein residues lead to the active site, forming hydrogen bonded "proton wires" that link general acid D379 with bulk solvent. These proton wires may facilitate proton transfer with the general acid during catalysis. Activity measurements made with mutant enzymes indicate that N425, which donates a hydrogen bond directly to D379, and T421, which hydrogen bonds with D379 through an intervening solvent molecule, help orient D379 for catalysis. Residues involved in hydrogen bonds with the proton wire, R340 and D503, are also important. Finally, conserved residue E211, which is located near the diphosphate group of 1, is proposed to be a ligand to Mg2 + required for optimal catalytic activity. General significance This work establishes structure-function relationships for class II terpenoid cyclases.
URI: https://doi.org/10.1016/j.bbagen.2013.09.004
http://hdl.handle.net/11147/5650
ISSN: 0304-4165
0304-4165
Appears in Collections:Molecular Biology and Genetics / Moleküler Biyoloji ve Genetik
PubMed İndeksli Yayınlar Koleksiyonu / PubMed Indexed Publications Collection
Scopus İndeksli Yayınlar Koleksiyonu / Scopus Indexed Publications Collection
WoS İndeksli Yayınlar Koleksiyonu / WoS Indexed Publications Collection

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