Iterative approach to computational enzyme design

Abstract

A general approach for the computational design of enzymes to catalyze arbitrary reactions is a goal at the forefront of the field of protein design. Recently, computationally designed enzymes have been produced for three chemical reactions through the synthesis and screening of a large number of variants. Here, we present an iterative approach that has led to the development of the most catalytically efficient computationally designed enzyme for the Kemp elimination to date. Previously established computational techniques were used to generate an initial design, HG-1, which was catalytically inactive. Analysis of HG-1 with molecular dynamics simulations (MD) and X-ray crystallography indicated that the inactivity might be due to bound waters and high flexibility of residues within the active site. This analysis guided changes to our design procedure, moved the design deeper into the interior of the protein, and resulted in an active Kemp eliminase, HG-2. The cocrystal structure of this enzyme with a transition state analog (TSA) revealed that the TSA was bound in the active site, interacted with the intended catalytic base in a catalytically relevant manner, but was flipped relative to the design model. MD analysis of HG-2 led to an additional point mutation, HG-3, that produced a further threefold improvement in activity. This iterative approach to computational enzyme design, including detailed MD and structural analysis of both active and inactive designs, promises a more complete understanding of the underlying principles of enzymatic catalysis and furthers progress toward reliably producing active enzymes.

Fig. 1.

The KE reaction scheme.

Fig. 2.

KE enzyme design models and crystal structures. (A) KE idealized active site. (B) Overlay of HG-1 crystal structure active site residues (yellow) with design model (green). (C) The HG-2 design model. (D) and (E) Crystal structure of HG-2 active site, chain A. The two conformations of the TSA 5-NBT are shown separately for clarity. (F) Crystal structure of HG-2 active site, chain B with the single observed conformation of the TSA.

Fig. 3.

MD-assisted design refinement: from HG-1 to HG-2. (A) and (B) MD base-substrate distance versus time plots (Asp OD to acidic hydrogen of 5-NBZ) for HG-1 (A) and HG-2 (B). (C) and (D) Angle versus distance scatter plots of the catalytic contact (as displayed in the Inset). Data points were taken from 20-ns MD trajectories of HG-2 with 5-NBZ bound in orientations O1 (C) and O2 (D). The coordinates of the TS geometry are displayed in filled discs.

Fig. 4.

Relative active site locations in the TAX scaffold and the designs. (A) The locations of the active sites of HG-1 (magenta mesh) and HG-2 (cyan mesh) in the TAX scaffold. The active sites of (B) the wild-type TAX scaffold, (C) the design model of HG-1, and (D) the design model of HG-2. The TS model in the two designs is shown in orange.

Fig. 5.

Kinetic characterization of Kemp elimination enzymes. Michaelis-Menten plots of HG-2 and point mutants. The enzyme concentration for all reactions was 5 μM.

Fig. 6.

Crystal structures of 1A53-2. (A) Overlay of 1A53-2 holostructure (yellow) and the design model (green). (B) Overlay of 1A53-2 apo crystal structure (lavender) and holostructure (yellow).