Automated Design of Efficient and Functionally Diverse Enzyme Repertoires

Abstract

Substantial improvements in enzyme activity demand multiple mutations at spatially proximal positions in the active site. Such mutations, however, often exhibit unpredictable epistatic (non-additive) effects on activity. Here we describe FuncLib, an automated method for designing multipoint mutations at enzyme active sites using phylogenetic analysis and Rosetta design calculations. We applied FuncLib to two unrelated enzymes, a phosphotriesterase and an acetyl-CoA synthetase. All designs were active, and most showed activity profiles that significantly differed from the wild-type and from one another. Several dozen designs with only 3-6 active-site mutations exhibited 10- to 4,000-fold higher efficiencies with a range of alternative substrates, including hydrolysis of the toxic organophosphate nerve agents soman and cyclosarin and synthesis of butyryl-CoA. FuncLib is implemented as a web server (http://FuncLib.weizmann.ac.il); it circumvents iterative, high-throughput experimental screens and opens the way to designing highly efficient and diverse catalytic repertoires.

Keywords: FuncLib; PROSS; Rosetta; enzyme design; enzyme repertoires; epistatic mutations; nerve agents.

Figure 1. Flow chart illustrating key steps in the design of functional repertoires.

The images illustrate steps in the generation of a repertoire of phosphotriesterase enzymes starting from a bacterial phosphotriesterase (PTE; PDB entry: 1HZY). (A) Active-site positions are selected for design, and at each position, sequence space is constrained by evolutionary-conservation analysis (PSSM) and mutational-scanning calculations (ΔΔG). (B) Multipoint mutants are exhaustively enumerated using Rosetta atomistic design calculations. The PTE active site comprises a bimetal center (gray spheres) of Zn²⁺ ions that are liganded by six highly conserved residues (gray sticks); eight additional residues (colored sticks) comprise the active-site wall and are less conserved. (C) The designs are ranked by energy, and (D) the sequences are clustered to obtain a repertoire of diverse, low-energy designs for experimental testing. Designed positions are colored consistently in all panels. See also Table S1.

Figure 2. A designed repertoire of phosphotriesterases exhibits orders of magnitude improvement in a range of promiscuous activities, including the hydrolysis of highly toxic nerve agents.

(A) The bacterial PTE is a paraoxonase exhibiting additional promiscuous hydrolase activities. The dashed red lines indicate the bonds that PTE hydrolyses in each of the substrates tested in this study, and the asterisks indicate chiral centers. (B) Fold improvement in catalytic efficiency (k_cat/K_M) of the top FuncLib designs relative to PTE-S5, showing remarkable >1,000-fold improvement in nerve-agent hydrolysis efficiency in several designs. The number of active-site mutations is indicated above the bars. (C) Activity profiles of the top PTE designs. Several designs, most prominently PTE_27, PTE_28, and PTE_27.14, exhibit substantially broadened substrate selectivity relative to PTE S5. Data for nerve agents are shown for the more toxic S_p stereoisomers. Data are represented as mean ± standard deviations of duplicate measurements. N.D. - not determined. See also Tables S1-S3.

Figure 3. The stereochemical properties of the designed active-site pockets underlie selectivity changes in PTE designs.

PTE_27 and PTE_28 exhibit a larger active-site pocket than PTE and high catalytic efficiency against bulky V- and G-type nerve agents. In clockwise order from top-left, molecular renderings are based on PDB entries: 1HZY, 6GBJ, 6GBK, and 6GBL (see Table S4). Spheres indicate ions of the bimetal center. Figure S2 shows models of the designed enzymes with docked substrates. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Molecular_Cell:2

Figure 4. Designed mutations exhibit sign-epistatic relationships.

Each green circle represents a mutant of dPTE2, and the gray orbits represent the number of mutations from dPTE2. The area of each circle is proportional to the design’s specific activity in hydrolyzing the aryl ester 2-naphthyl acetate (2NA). PROSS-stabilized design dPTE2, which was used as the starting point for FuncLib design, exhibits low specific activity. Each of the point mutants exhibits improved specific activity, but specific activity declines in the double mutants. Last, the quad-mutant, design PTE_5, substantially improves specific activity relative to all single or double mutants. See also Figure S3.

Figure 5. A designed repertoire of acyl-CoA-synthetases and substantially broadened substrate selectivity.

(A) The ACS active site. Positions where design was enabled are shown as green sticks, and AMP and CoA substrates are shown as teal lines. Amino acid positions and identities allowed during design are indicated. (B) Specific activity of ACS variants, normalized to that of ACS_PROSS with acetate, demonstrates substantial broadening of substrate selectivity in designs ACS_9, ACS_16, and ACS_27. See also Tables S5-S6 and Figures S4-S5.