Structure-Guided Triple-Code Saturation Mutagenesis: Efficient Tuning of the Stereoselectivity of an Epoxide Hydrolase

Abstract

he directed evolution of enzymes promises to eliminate the long-standing limitations of biocatalysis in organic chemistry and biotechnology-the often-observed limited substrate scope, insufficient activity, and poor regioselectivity or stereoselectivity. Saturation mutagenesis at sites lining the binding pocket with formation of focused libraries has emerged as the technique of choice, but choosing the optimal size of the randomization site and reduced amino acid alphabet for minimizing the labor-determining screening effort remains a challenge. Here, we introduce structure-guided triple-code saturation mutagenesis (TCSM) by encoding three rationally chosen amino acids as building blocks in the randomization of large multiresidue sites. In contrast to conventional NNK codon degeneracy encoding all 20 canonical amino acids and requiring the screening of more than 10(15) transformants for 95% library coverage, TCSM requires only small libraries not exceeding 200800 transformants in one library. The triple code utilizes structural (X-ray) and consensus-derived sequence data, and is therefore designed to match the steric and electrostatic characteristics of the particular enzyme. Using this approach, limonene epoxide hydrolase has been successfully engineered as stereoselective catalysts in the hydrolytic desymmetrization of meso-type epoxides with formation of either (R,R)- or (S,S)-configurated diols on an optional basis and kinetic resolution of chiral substrates. Crystal structures and docking computations support the source of notably enhanced and inverted enantioselectivity

Exploring productive sequence space in directed evolution using binary patterning versus conventional mutagenesis strategies

“…Given that most of the residues lining the LEH binding pocket are hydrophobic (van der Werf et al 1998;Arand et al 2003), this may not be surprising. Indeed, it supports our previous conjectures and choices of reduced amino acids in the directed evolution of LEH (Sun et al 2015(Sun et al , 2016b. However, choosing only hydrophobic amino acids as components of reduced amino acid alphabets cannot be viewed as a general guideline.…”

“…In previous LEH studies describing the hydrolytic desymmetrization of cyclohexene oxide (1) with formation of (R,R)-and (S,S)-2 (Zheng and Reetz 2010;Sun et al 2015Sun et al , 2016b, saturation mutagenesis was focused on sites lining the binding pocket by employing several different approaches, including single code saturation mutagenesis (SCSM) (Sun et al 2015), double code saturation mutagenesis (DCSM) (Sun et al 2016c) and triple code saturation mutagenesis (TCSM) (Sun et al 2016b) as extensions of the CASTing tool box. New mutants were discovered independently, displaying excellent enantioselectivity.…”

“…Similar to previous directed evolution studies of LEH (Sun et al 2015(Sun et al , 2016b, the crystal structure of wildtype (WT) LEH (Arand et al 2003) served as a guide for choosing CAST residues. In the present study, all together eight residues in close proximity of the active site of LEH (PDB 1NU3) were selected for SM, which were grouped in two randomization locations, sites 1 (I80, V83, L114 and I116) and site 2 (L35, L74, L103 and F134).…”

“…For several years we have employed limonene epoxide hydrolase (LEH) (van der Werf et al 1998;Arand et al 2003) as the model reaction for methodology development in directed evolution, the hydrolytic desymmetrization of cyclohexene oxide (1) with formation of (R,R)-and (S,S)-2 serving as the model reaction (Scheme 1) (Zheng and Reetz 2010;Sun et al 2015Sun et al , 2016b. Various reduced amino acid alphabets were tested, triple code saturation mutagenesis (TCSM) encoding three properly chosen amino acids as building blocks (in addition to wildtype, WT) appearing to be a reasonable compromise between structural diversity and degree of screening (Sun et al 2016b).…”

“…Various reduced amino acid alphabets were tested, triple code saturation mutagenesis (TCSM) encoding three properly chosen amino acids as building blocks (in addition to wildtype, WT) appearing to be a reasonable compromise between structural diversity and degree of screening (Sun et al 2016b). In the present study we considered binary patterning in designing SM libraries and compared them to conventional SM libraries.…”