6AXU

W203Y Epi-isozizaene synthase

6AXU の概要

• エントリーDOI: 10.2210/pdb6axu/pdb
• 分子名称: Epi-isozizaene synthase, N-benzyl-N,N-diethylethanaminium, PYROPHOSPHATE 2-, ... (5 entities in total)
• 機能のキーワード: terpenoid cyclase, lyase
• 由来する生物種: Streptomyces coelicolor
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 44134.17

構造登録者

Blank, P.N.,Barrow, G.H.,Christianson, D.W. (登録日: 2017-09-07, 公開日: 2017-10-18, 最終更新日: 2023-10-04)

主引用文献

Blank, P.N.,Barrow, G.H.,Chou, W.K.W.,Duan, L.,Cane, D.E.,Christianson, D.W.
Substitution of Aromatic Residues with Polar Residues in the Active Site Pocket of epi-Isozizaene Synthase Leads to the Generation of New Cyclic Sesquiterpenes.
Biochemistry, 56:5798-5811, 2017

PubMed Abstract: The sesquiterpene cyclase epi-isozizaene synthase (EIZS) catalyzes the cyclization of farnesyl diphosphate to form the tricyclic hydrocarbon precursor of the antibiotic albaflavenone. The hydrophobic active site pocket of EIZS serves as a template as it binds and chaperones the flexible substrate and carbocation intermediates through the conformations required for a multistep reaction sequence. We previously demonstrated that the substitution of hydrophobic residues with other hydrophobic residues remolds the template and expands product chemodiversity [Li, R., Chou, W. K. W., Himmelberger, J. A., Litwin, K. M., Harris, G. G., Cane, D. E., and Christianson, D. W. (2014) Biochemistry 53, 1155-1168]. Here, we show that the substitution of hydrophobic residues-specifically, Y69, F95, F96, and W203-with polar side chains also yields functional enzyme catalysts that expand product chemodiversity. Fourteen new EIZS mutants are reported that generate product arrays in which eight new sesquiterpene products have been identified. Of note, some mutants generate acyclic and cyclic hydroxylated products, suggesting that the introduction of polarity in the hydrophobic pocket facilitates the binding of water capable of quenching carbocation intermediates. Furthermore, the substitution of polar residues for F96 yields high-fidelity sesquisabinene synthases. Crystal structures of selected mutants reveal that residues defining the three-dimensional contour of the hydrophobic pocket can be substituted without triggering significant structural changes elsewhere in the active site. Thus, more radical nonpolar-polar amino acid substitutions should be considered when terpenoid cyclase active sites are remolded by mutagenesis with the goal of exploring and expanding product chemodiversity.

PubMed: 28967743
DOI: 10.1021/acs.biochem.7b00895

実験手法

X-RAY DIFFRACTION (1.822 Å)