New algorithms and an in silico benchmark for computational enzyme design

Abstract

The creation of novel enzymes capable of catalyzing any desired chemical reaction is a grand challenge for computational protein design. Here we describe two new algorithms for enzyme design that employ hashing techniques to allow searching through large numbers of protein scaffolds for optimal catalytic site placement. We also describe an in silico benchmark, based on the recapitulation of the active sites of native enzymes, that allows rapid evaluation and testing of enzyme design methodologies. In the benchmark test, which consists of designing sites for each of 10 different chemical reactions in backbone scaffolds derived from 10 enzymes catalyzing the reactions, the new methods succeed in identifying the native site in the native scaffold and ranking it within the top five designs for six of the 10 reactions. The new methods can be directly applied to the design of new enzymes, and the benchmark provides a powerful in silico test for guiding improvements in computational enzyme design.

Figure 1.

Diagram of the computational enzyme design procedure.

Figure 2.

Energy distribution of native and non-native designed sites. (Left panel) The distribution of virtual energy and LJ repulsive energy between the TS model and the protein scaffold after minimizing the catalytic residues. (Right panel) The distribution of the computed catalytic efficacy (TS binding energy) after designing the binding pocket. The native matches (in the native scaffold in the native positions) are in black; the matches in alternate sites and/or scaffolds are in gray.

Figure 3.

Superposition of native and predicted active sites for fuculose 1-phosphate aldolase (4fua) and DERA aldolase (1jcl). Orange, native TS model position and catalytic side chains; green, designed TS model position and catalytic side chains. The TS model is represented with thick sticks; the catalytic side chains, with thin sticks.

Figure 4.

Grafting an aldolase active site onto a decarboxylase scaffold. The top panel is an overall view of the designed protein; the bottom panel is a closer view of the active site. The protein backbone is shown in cartoon mode and colored according to its secondary structure. The TS model is shown in ball-and-stick mode, and the TS analog carbon atoms are colored in orange. The designed catalytic residues (Lys, Asp, Lys) are shown as sticks, and their carbon atoms are colored in green.

Figure 5.

Illustration of geometric parameters used in active site description for deoxyribose-phosphate aldolase (DERA). Six geometrical parameters (d indicates distance; θ₁ and θ₂ indicate bond angles; χ₁, χ₂, and χ₃ indicate torsional angles) are specified to describe the spatial positions of the functional groups relative to the TS.

Figure 6.

Inverse rotamer tree for deoxyribose-phosphate aldolase (DERA) active site. The transition state is colored in yellow, and the key functional groups of the catalytic residues are in gold. The remainder of the side chains in the rotamer trees are shown using thinner lines in CPK coloring.