Robert J. Floor

Structure- and sequence-analysis inspired engineering of proteins for enhanced thermostability

Protein engineering strategies for increasing stability can be improved by replacing random mutagenesis and high-throughput screening by approaches that include bioinformatics and computational design. Mutations can be focused on regions in the structure that are most flexible and involved in the early steps of thermal unfolding. Sequence analysis can often predict the position and nature of stabilizing mutations, and may allow the reconstruction of thermostable ancestral sequences. Various computational tools make it possible to design stabilizing features, such as hydrophobic clusters and surface charges. Different methods for designing chimeric enzymes can also support the engineering of more stable proteins without the need of high-throughput screening.

Computationally designed libraries for rapid enzyme stabilization

The ability to engineer enzymes and other proteins to any desired stability would have wide-ranging applications. Here, we demonstrate that computational design of a library with chemically diverse stabilizing mutations allows the engineering of drastically stabilized and fully functional variants of the mesostable enzyme limonene epoxide hydrolase. First, point mutations were selected if they significantly improved the predicted free energy of protein folding. Disulfide bonds were designed using sampling of backbone conformational space, which tripled the number of experimentally stabilizing disulfide bridges. Next, orthogonal in silico screening steps were used to remove chemically unreasonable mutations and mutations that are predicted to increase protein flexibility. The resulting library of 64 variants was experimentally screened, which revealed 21 (pairs of) stabilizing mutations located both in relatively rigid and in flexible areas of the enzyme. Finally, combining 10–12 of these confirmed mutations resulted in multi-site mutants with an increase in apparent melting temperature from 50 to 85°C, enhanced catalytic activity, preserved regioselectivity and a >250-fold longer half-life. The developed Framework for Rapid Enzyme Stabilization by Computational libraries (FRESCO) requires far less screening than conventional directed evolution.

Directed Evolution Strategies for Enantiocomplementary Haloalkane Dehalogenases: From Chemical Waste to Enantiopure Building Blocks

We used directed evolution to obtain enantiocomplementary haloalkane dehalogenase variants that convert the toxic waste compound 1,2,3‐trichloropropane (TCP) into highly enantioenriched (R)‐ or (S)‐2,3‐dichloropropan‐1‐ol, which can easily be converted into optically active epichlorohydrins—attractive intermediates for the synthesis of enantiopure fine chemicals. A dehalogenase with improved catalytic activity but very low enantioselectivity was used as the starting point. A strategy that made optimal use of the limited capacity of the screening assay, which was based on chiral gas chromatography, was developed. We used pair‐wise site‐saturation mutagenesis (SSM) of all 16 noncatalytic active‐site residues during the initial two rounds of evolution. The resulting best R‐ and S‐enantioselective variants were further improved in two rounds of site‐restricted mutagenesis (SRM), with incorporation of carefully selected sets of amino acids at a larger number of positions, including sites that are more distant from the active site. Finally, the most promising mutations and positions were promoted to a combinatorial library by using a multi‐site mutagenesis protocol with restricted codon sets. To guide the design of partly undefined (ambiguous) codon sets for these restricted libraries we employed structural information, the results of multiple sequence alignments, and knowledge from earlier rounds. After five rounds of evolution with screening of only 5500 clones, we obtained two strongly diverged haloalkane dehalogenase variants that give access to (R)‐epichlorohydrin with 90 % ee and to (S)‐epichlorohydrin with 97 % ee, containing 13 and 17 mutations, respectively, around their active sites.

Enantioselective enzymes by computational design and in silico screening.

Computational enzyme design holds great promise for providing new biocatalysts for synthetic chemistry. A strategy to design small mutant libraries of complementary enantioselective epoxide hydrolase variants for the production of highly enantioenriched (S,S)-diols and (R,R)-diols is developed. Key features of this strategy (CASCO, catalytic selectivity by computational design) are the design of mutations that favor binding of the substrate in a predefined orientation, the introduction of steric hindrance to prevent unwanted substrate binding modes, and ranking of designs by high-throughput molecular dynamics simulations. Using this strategy we obtained highly stereoselective mutants of limonene epoxide hydrolase after experimental screening of only 37 variants. The results indicate that computational methods can replace a substantial amount of laboratory work when developing enantioselective enzymes.

Biocatalytic and structural properties of a highly engineered halohydrin dehalogenase.

Two highly engineered halohydrin dehalogenase variants were characterized in terms of their performance in dehalogenation and epoxide cyanolysis reactions. Both enzyme variants outperformed the wild‐type enzyme in the cyanolysis of ethyl (S)‐3,4‐epoxybutyrate, a conversion yielding ethyl (R)‐4‐cyano‐3‐hydroxybutyrate, an important chiral building block for statin synthesis. One of the enzyme variants, HheC2360, displayed catalytic rates for this cyanolysis reaction enhanced up to tenfold. Furthermore, the enantioselectivity of this variant was the opposite of that of the wild‐type enzyme, both for dehalogenation and for cyanolysis reactions. The 37‐fold mutant HheC2360 showed an increase in thermal stability of 8 °C relative to the wild‐type enzyme. Crystal structures of this enzyme were elucidated with chloride and ethyl (S)‐3,4‐epoxybutyrate or with ethyl (R)‐4‐cyano‐3‐hydroxybutyrate bound in the active site. The observed increase in temperature stability was explained in terms of a substantial increase in buried surface area relative to the wild‐type HheC, together with enhanced interfacial interactions between the subunits that form the tetramer. The structures also revealed that the substrate binding pocket was modified both by substitutions and by backbone movements in loops surrounding the active site. The observed changes in the mutant structures are partly governed by coupled mutations, some of which are necessary to remove steric clashes or to allow backbone movements to occur. The importance of interactions between substitutions suggests that efficient directed evolution strategies should allow for compensating and synergistic mutations during library design.

Computational Library Design for Increasing Haloalkane Dehalogenase Stability

We explored the use of a computational design framework for the stabilization of the haloalkane dehalogenase LinB. Energy calculations, disulfide bond design, molecular dynamics simulations, and rational inspection of mutant structures predicted many stabilizing mutations. Screening of these in small mutant libraries led to the discovery of seventeen point mutations and one disulfide bond that enhanced thermostability. Mutations located in or contacting flexible regions of the protein had a larger stabilizing effect than mutations outside such regions. The combined introduction of twelve stabilizing mutations resulted in a LinB mutant with a 23 °C increase in apparent melting temperature (Tm,app, 72.5 °C) and an over 200‐fold longer half‐life at 60 °C. The most stable LinB variants also displayed increased compatibility with co‐solvents, thus allowing substrate conversion and kinetic resolution at much higher concentrations than with the wild‐type enzyme.

Hydrolysis and Reverse Hydrolysis : Halohydrin Dehalogenases

Halohydrin dehalogenases are members of a distinct class of enzymes that catalyze the release of halides from vicinal halohydrins with formation of the corresponding epoxides. In the reverse direction, these enzymes also catalyze the nucleophilic epoxide ring-opening with alternative anions such as cyanide, azide, and nitrite, yielding important β -substituted alcohols. Some of these reactions proceed with excellent regio- and enantioselectivity, making halohydrin dehalogenases interesting biocatalysts for synthetic chemistry applications. This chapter gives an overview of mechanistic and engineering studies of these enzymes. Further, examples of biocatalytic conversions are presented which show the usefulness and versatility of these dehalogenases in the preparation of intermediates for enantiopure fine chemicals.

A robust cosolvent-compatible halohydrin dehalogenase by computational library design

To improve the applicability of halohydrin dehalogenase as a catalyst for reactions in the presence of organic cosolvents, we explored a computational library design strategy (Framework for Rapid Enzyme Stabilization by Computational libraries) that involves discovery and in silico evaluation of stabilizing mutations. Energy calculations, disulfide bond predictions and molecular dynamics simulations identified 218 point mutations and 35 disulfide bonds with predicted stabilizing effects. Experiments confirmed 29 stabilizing point mutations, most of which were located in two distinct regions, whereas introduction of disulfide bonds was not effective. Combining the best mutations resulted in a 12-fold mutant (HheC-H12) with a 28°C higher apparent melting temperature and a remarkable increase in resistance to cosolvents. This variant also showed a higher optimum temperature for catalysis while activity at low temperature was preserved. Mutant H12 was used as a template for the introduction of mutations that enhance enantioselectivity or activity. Crystal structures showed that the structural changes in the H12 mutant mostly agreed with the computational predictions and that the enhanced stability was mainly due to mutations that redistributed surface charges and improved interactions between subunits, the latter including better interactions of water molecules at the subunit interfaces.

X‐ray crystallographic validation of structure predictions used in computational design for protein stabilization

Protein engineering aimed at enhancing enzyme stability is increasingly supported by computational methods for calculation of mutant folding energies and for the design of disulfide bonds. To examine the accuracy of mutant structure predictions underlying these computational methods, crystal structures of thermostable limonene epoxide hydrolase variants obtained by computational library design were determined. Four different predicted effects indeed contributed to the obtained stabilization: (i) enhanced interactions between a flexible loop close to the N‐terminus and the rest of the protein; (ii) improved interactions at the dimer interface; (iii) removal of unsatisfied hydrogen bonding groups; and (iv) introduction of additional positively charged groups at the surface. The structures of an eightfold and an elevenfold mutant showed that most mutations introduced the intended stabilizing interactions, and side‐chain conformations were correctly predicted for 72–88% of the point mutations. However, mutations that introduced a disulfide bond in a flexible region had a larger influence on the backbone conformation than predicted. The enzyme active sites were unaltered, in agreement with the observed preservation of catalytic activities. The structures also revealed how a c‐Myc tag, which was introduced for facile detection and purification, can reduce access to the active site and thereby lower the catalytic activity. Finally, sequence analysis showed that comprehensive mutant energy calculations discovered stabilizing mutations that are not proposed by the consensus or B‐FIT methods. Proteins 2015; 83:940–951.