3TC6

Crystal Structure of Engineered Protein. Northeast Structural Genomics Consortium Target OR63.

3TC6 の概要

• エントリーDOI: 10.2210/pdb3tc6/pdb
• 分子名称: Indole-3-glycerol phosphate synthase, PHOSPHATE ION, DI(HYDROXYETHYL)ETHER, ... (4 entities in total)
• 機能のキーワード: structural genomics, engineered protein, northeast structural genomics consortium, nesg, or63, psi-biology, lyase
• 由来する生物種: Sulfolobus solfataricus P2
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 30245.25

構造登録者

Vorobiev, S.,Su, M.,Bjelic, S.,Kipnis, Y.,Wang, L.,Seetharaman, J.,Sahdev, S.,Xiao, R.,Ciccosanti, C.,Baker, D.,Everett, J.K.,Acton, T.B.,Montelione, G.T.,Hunt, J.F.,Tong, L.,Northeast Structural Genomics Consortium (NESG) (登録日: 2011-08-08, 公開日: 2011-08-24, 最終更新日: 2023-09-13)

主引用文献

Bjelic, S.,Kipnis, Y.,Wang, L.,Pianowski, Z.,Vorobiev, S.,Su, M.,Seetharaman, J.,Xiao, R.,Kornhaber, G.,Hunt, J.F.,Tong, L.,Hilvert, D.,Baker, D.
Exploration of alternate catalytic mechanisms and optimization strategies for retroaldolase design.
J.Mol.Biol., 426:256-271, 2014

PubMed Abstract: Designed retroaldolases have utilized a nucleophilic lysine to promote carbon-carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (>10(-3)s(-1)) and kcat/KM (11-25M(-1)s(-1)) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the >10(5)-fold rate accelerations that were achieved are within 1-3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat=10(6) to 10(8)) and an extensively evolved computational design (kcat/kuncat>10(7)). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches.

PubMed: 24161950
DOI: 10.1016/j.jmb.2013.10.012

実験手法

X-RAY DIFFRACTION (1.6 Å)