3TC7

Crystal Structure of Engineered Protein. Northeast Structural Genomics Consortium Target OR62.

Summary for 3TC7

• Entry DOI: 10.2210/pdb3tc7/pdb
• Related: 3TC6
• Descriptor: Indole-3-glycerol phosphate synthase, PHOSPHATE ION, ACETIC ACID, ... (4 entities in total)
• Functional Keywords: engineered protein, protein structure initiative, northeast structural genomics consortium, nesg, or62, psi-biology, lyase
• Biological source: Sulfolobus solfataricus P2
• Total number of polymer chains: 1
• Total formula weight: 30142.18

Authors

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) (deposition date: 2011-08-08, release date: 2011-08-24, Last modification date: 2023-09-13)

Primary citation

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

Experimental method

X-RAY DIFFRACTION (1.5 Å)