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J Mol Biol. 2012 Jan 20;415(3):615-25. doi: 10.1016/j.jmb.2011.10.043. Epub 2011 Nov 3.

Structural analyses of covalent enzyme-substrate analog complexes reveal strengths and limitations of de novo enzyme design.

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1
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.

Abstract

We report the cocrystal structures of a computationally designed and experimentally optimized retro-aldol enzyme with covalently bound substrate analogs. The structure with a covalently bound mechanism-based inhibitor is similar to, but not identical with, the design model, with an RMSD of 1.4 Å over active-site residues and equivalent substrate atoms. As in the design model, the binding pocket orients the substrate through hydrophobic interactions with the naphthyl moiety such that the oxygen atoms analogous to the carbinolamine and β-hydroxyl oxygens are positioned near a network of bound waters. However, there are differences between the design model and the structure: the orientation of the naphthyl group and the conformation of the catalytic lysine are slightly different; the bound water network appears to be more extensive; and the bound substrate analog exhibits more conformational heterogeneity than typical native enzyme-inhibitor complexes. Alanine scanning of the active-site residues shows that both the catalytic lysine and the residues around the binding pocket for the substrate naphthyl group make critical contributions to catalysis. Mutating the set of water-coordinating residues also significantly reduces catalytic activity. The crystal structure of the enzyme with a smaller substrate analog that lacks naphthyl ring shows the catalytic lysine to be more flexible than in the naphthyl-substrate complex; increased preorganization of the active site would likely improve catalysis. The covalently bound complex structures and mutagenesis data highlight the strengths and weaknesses of the de novo enzyme design strategy.

PMID:
22075445
PMCID:
PMC3440004
DOI:
10.1016/j.jmb.2011.10.043
[Indexed for MEDLINE]
Free PMC Article
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