Design of activated serine–containing catalytic triads with atomic-level accuracy

Abstract

A challenge in the computational design of enzymes is that multiple properties must be simultaneously optimized -- substrate-binding, transition state stabilization, and product release -- and this has limited the absolute activity of successful designs. Here, we focus on a single critical property of many enzymes: the nucleophilicity of an active site residue that initiates catalysis. We design proteins with idealized serine-containing catalytic triads, and assess their nucleophilicity directly in native biological systems using activity-based organophosphate probes. Crystal structures of the most successful designs show unprecedented agreement with computational models, including extensive hydrogen bonding networks between the catalytic triad (or quartet) residues, and mutagenesis experiments demonstrate that these networks are critical for serine activation and organophosphate-reactivity. Following optimization by yeast-display, the designs react with organophosphate probes at rates comparable to natural serine hydrolases. Co-crystal structures with diisopropyl fluorophosphate bound to the serine nucleophile suggest the designs could provide the basis for a new class of organophosphate captures agents.

Advances in the directed evolution of proteins

“…In a separate effort to create a toxin-neutralizing protein, a unique catalytic triad was computationally designed and subsequently optimized by yeast display. The resulting protein reacted with a fluorophosphonate probe at rates comparable to natural serine hydrolases, yet it was incapable of catalytic turnover [67]. …”

Installing hydrolytic activity into a completely de novo protein framework

“…The designs are selected through computational modeling of transition-state analogues within side-chain constellations. 7,8 Iterative rounds of computational design, both in the active site and surrounding residues, place mutations to stabilize the transition state with the aim of accelerating the target reaction. Successes of this approach include redesigned proteins with activities for the Kemp elimination, 9 and the retroaldol 10 and Diels-Alder 11 reactions.…”

Harnessing Nature’s Diversity: Discovering organophosphate bioscavenger characteristics among low molecular weight proteins

“…Recent studies by Steinkellner et al have identified two proteins with previously unknown promiscuous ene-reductase activity through geometric comparison13. Whereas, Rajagopalan et al grafts a desired amino acid structure by de novo design protocols to create novel serine esterases14.…”