Structural Analyses of Covalent Enzyme-Substrate Analog Complexes Reveal the Strengths and Limitations of De Novo Enzyme Design.

Publication Type:

Journal Article


Journal of molecular biology, Volume 415, Issue 3, p.615-625 (2012)


2012, Amino Acid Substitution, Basic Sciences Division, CATALYTIC DOMAIN, Center-Authored Paper, Crystallography, X-Ray, December 2011, Enzyme Inhibitors, Fructose-Bisphosphate Aldolase, Models, Molecular, Mutagenesis, Site-Directed, Mutant Proteins, Protein Engineering


We report the cocrystal structures of a computationally designed and experimentally optimized retro-aldol enzyme with covalently bound substrate analogs. The structure with covalently bound substrate analogs 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 rings 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.