Xu-Dong Kong

Reshaping an Enzyme Binding Pocket for Enhanced and Inverted Stereoselectivity: Use of Smallest Amino Acid Alphabets in Directed Evolution

Directed evolution based on saturation mutagenesis at sites lining the binding pocket is a commonly practiced strategy for enhancing or inverting the stereoselectivity of enzymes for use in organic chemistry or biotechnology. However, as the number of residues in a randomization site increases to five or more, the screening effort for 95 % library coverage increases astronomically until it is no longer feasible. We propose the use of a single amino acid for saturation mutagenesis at superlarge randomization sites comprising 10 or more residues. When used to reshape the binding pocket of limonene epoxide hydrolase, this strategy, which drastically reduces the search space and thus the screening effort, resulted in R,R- and S,S-selective mutants for the hydrolytic desymmetrization of cyclohexene oxide and other epoxides. X-ray crystal structures and docking studies of the mutants unveiled the source of stereoselectivity and shed light on the mechanistic intricacies of this enzyme.

Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates

Significance Application of epoxide hydrolases in synthesizing chiral drug compounds has been hindered by their limited substrate range. The enzymatic production of bulky epoxides has proven remarkably challenging. In this work, we identified an active tunnel for substrate access and product release of an epoxide hydrolase with unusual (R)-enantioselectivity. Mutagenesis targeted to unblock the steric hindrance in the active pocket or the potential product release site resulted in variants with much higher activity toward α-naphthyl glycidyl ether, the precursor of β-adrenergic receptor blocking drug (S)-propranolol. The strategy presented here may be a useful alternative choice for rational design of enzymes toward bulky substrates. Optically pure epoxides are essential chiral precursors for the production of (S)-propranolol, (S)-alprenolol, and other β-adrenergic receptor blocking drugs. Although the enzymatic production of these bulky epoxides has proven difficult, here we report a method to effectively improve the activity of BmEH, an epoxide hydrolase from Bacillus megaterium ECU1001 toward α-naphthyl glycidyl ether, the precursor of (S)-propranolol, by eliminating the steric hindrance near the potential product-release site. Using X-ray crystallography, mass spectrum, and molecular dynamics calculations, we have identified an active tunnel for substrate access and product release of this enzyme. The crystal structures revealed that there is an independent product-release site in BmEH that was not included in other reported epoxide hydrolase structures. By alanine scanning, two mutants, F128A and M145A, targeted to expand the potential product-release site displayed 42 and 25 times higher activities toward α-naphthyl glycidyl ether than the wild-type enzyme, respectively. These results show great promise for structure-based rational design in improving the catalytic efficiency of industrial enzymes for bulky substrates.

A Smart Library of Epoxide Hydrolase Variants and the Top Hits for Synthesis of (S)‐β‐Blocker Precursors

Microtuning of the enzyme active pocket has led to a smart library of epoxide hydrolase variants with an expanded substrate spectrum covering a series of typical β-blocker precursors. Improved activities of 6- to 430-fold were achieved by redesigning the active site at two predicted hot spots. This study represents a breakthrough in protein engineering of epoxide hydrolases and resulted in enhanced activity toward bulky substrates.

Switching a newly discovered lactonase into an efficient and thermostable phosphotriesterase by simple double mutations His250Ile/Ile263Trp.

OPHC2 is a thermostable organophosphate (OP) hydrolase in the β‐lactamase superfamily. OPs are highly toxic synthetic chemicals with no natural analogs. How did OPHC2 acquire phosphotriesterase (PTE) activity remained unclear. In this study, an OPHC2 analogue, PoOPH was discovered from Pseudomonas oleovorans exhibiting high lactonase and esterase activities and latent PTE activity. Sequence analysis revealed conserved His250 and Ile263 and site‐directed mutagenesis at these crucial residues enhanced PTE activity. The best variant PoOPHM2 carrying H250I/I263W mutations displayed 6,962‐ and 106‐fold improvements in catalytic efficiency for methyl‐parathion and ethyl‐paraoxon degradation, whereas the original lactonase and esterase activities decreased dramatically. A 1.4 × 107‐fold of specificity inversion was achieved by only two residue substitutions. Significantly, thermostability of the variants was not compromised. Crystal structure of PoOPHM2 was determined at 2.25 Å resolution and docking studies suggested that the two residues in the binding pocket determine substrate recognition. Lastly, new organophosphorus hydrolases (OPHs) were discovered using simple double mutations. Among them, PpOPHM2 from Pseudomonas putida emerged as a new promising OPH with very high activity (41.0 U mg−1) toward methyl‐parathion. Our results offer a first scrutiny to PTE activity evolution of OPHs in β‐lactamase superfamily and provide efficient and robust enzymes for OP detoxification. Biotechnol. Bioeng. 2014;111: 1920–1930.

Substrate channel evolution of an esterase for the synthesis of cilastatin

The esterase RhEst1 from Rhodococcus sp. ECU1013 has been reported for the enantioselective hydrolysis of ethyl (S)-(+)-2,2-dimethylcyclopropane carboxylate, producing the building block of cilastatin. In this work, error-prone PCR and site-directed saturation mutagenesis were applied to RhEst1 for activity improvement, with the pH-indicator assay as a high-throughput screening method. As a result, RhEst1A147I/V148F/G254A, with mutations surrounding the substrate access channel, showed a 5-fold increase in its specific activity compared with the native enzyme, as well as a 4-fold increase in protein solubility. Combined with the determination of protein structures and computational analysis, this work shows that the amino acids around the substrate channel play a more important role in the activity evolution of RhEst1 than those in the active site.

Efficient synthesis of a statin precursor in high space-time yield by a new aldehyde-tolerant aldolase identified from Lactobacillus brevis

A novel 2-deoxyribose-5-phosphate aldolase (LbDERA) was identified from Lactobacillus brevis, with high activity, excellent thermostability and high tolerance against aldehyde substrates. The half-lives of LbDERA incubated in 300 mM acetaldehyde and chloroacetaldehyde were 37.3 and 198 min, respectively, which are 2- and 7-fold higher than those of EcDERA from Escherichia coli. The crystal structure of LbDERA determined at 1.95 A resolution revealed a stable quaternary structure which might account for its excellent aldehyde tolerance. A single mutation, E78K, was introduced to LbDERA through a consensus sequence approach, resulting in significant improvements of both thermostability and aldehyde tolerance. According to the crystal structure of LbDERAE78K, two additional hydrogen bonds and one salt bridge were introduced compared with wild-type LbDERA. As a result of its high substrate tolerance, LbDERAE78K could efficiently catalyze a sequential aldol condensation with 0.7 M chloroacetaldehyde and 1.4 M acetaldehyde, affording a key chiral precursor of statins, (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside, with an unprecedented space-time yield of 792.5 g L−1 d−1 and only 2.5 g L−1 of catalyst loading.

Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

ABSTRACT Streptomyces coelicolor CR1 (ScCR1) has been shown to be a promising biocatalyst for the synthesis of an atorvastatin precursor, ethyl-(S)-4-chloro-3-hydroxybutyrate [(S)-CHBE]. However, limitations of ScCR1 observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. First, the crystal structure of ScCR1 complexed with NADH and cosubstrate 2-propanol was solved, and the specific activity of ScCR1 was increased from 38.8 U/mg to 168 U/mg (ScCR1I158V/P168S) by structure-guided engineering. Second, directed evolution was performed to improve the stability using ScCR1I158V/P168S as a template, affording a triple mutant, ScCR1A60T/I158V/P168S, whose thermostability (T5015, defined as the temperature at which 50% of initial enzyme activity is lost following a heat treatment for 15 min) and substrate tolerance (C5015, defined as the concentration at which 50% of initial enzyme activity is lost following incubation for 15 min) were 6.2°C and 4.7-fold higher than those of the wild-type enzyme. Interestingly, the specific activity of the triple mutant was further increased to 260 U/mg. Protein modeling and docking analysis shed light on the origin of the improved activity and stability. In the asymmetric reduction of ethyl-4-chloro-3-oxobutyrate (COBE) on a 300-ml scale, 100 g/liter COBE could be completely converted by only 2 g/liter of lyophilized ScCR1A60T/I158V/P168S within 9 h, affording an excellent enantiomeric excess (ee) of >99% and a space-time yield of 255 g liter−1 day−1. These results suggest high efficiency of the protein engineering strategy and good potential of the resulting variant for efficient synthesis of the atorvastatin precursor. IMPORTANCE Application of the carbonyl reductase ScCR1 in asymmetrically synthesizing (S)-CHBE, a key precursor for the blockbuster drug Lipitor, from COBE has been hindered by its low catalytic activity and poor thermostability and substrate tolerance. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. The catalytic efficiency, thermostability, and substrate tolerance of ScCR1 were significantly improved by structure-guided engineering and directed evolution. The engineered ScCR1 may serve as a promising biocatalyst for the biosynthesis of (S)-CHBE, and the protein engineering strategy adopted in this work would serve as a useful approach for future engineering of other reductases toward potential application in organic synthesis.

Structural and Computational Insight into the Catalytic Mechanism of Limonene Epoxide Hydrolase Mutants in Stereoselective Transformations.

Directed evolution of limonene epoxide hydrolase (LEH), which catalyzes the hydrolytic desymmetrization reactions of cyclopentene oxide and cyclohexene oxide, results in (R,R)- and (S,S)-selective mutants. Their crystal structures combined with extensive theoretical computations shed light on the mechanistic intricacies of this widely used enzyme. From the computed activation energies of various pathways, we discover the underlying stereochemistry for favorable reactions. Surprisingly, some of the most enantioselective mutants that rapidly convert cyclohexene oxide do not catalyze the analogous transformation of the structurally similar cyclopentene oxide, as shown by additional X-ray structures of the variants harboring this slightly smaller substrate. We explain this puzzling observation on the basis of computational calculations which reveal a disrupted alignment between nucleophilic water and cyclopentene oxide due to the pronounced flexibility of the binding pocket. In contrast, in the stereoselective reactions of cyclohexene oxide, reactive conformations are easily reached. The unique combination of structural and computational data allows insight into mechanistic details of this epoxide hydrolase and provides guidance for future protein engineering in reactions of structurally different substrates.

Crystal structure of tyrosine decarboxylase and identification of key residues involved in conformational swing and substrate binding.

Tyrosine decarboxylase (TDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme and is mainly responsible for the synthesis of tyramine, an important biogenic amine. In this study, the crystal structures of the apo and holo forms of Lactobacillus brevis TDC (LbTDC) were determined. The LbTDC displays only 25% sequence identity with the only reported TDC structure. Site-directed mutagenesis of the conformationally flexible sites and catalytic center was performed to investigate the potential catalytic mechanism. It was found that H241 in the active site plays an important role in PLP binding because it has different conformations in the apo and holo structures of LbTDC. After binding to PLP, H241 rotated to the position adjacent to the PLP pyridine ring. Alanine scanning mutagenesis revealed several crucial regions that determine the substrate specificity and catalytic activity. Among the mutants, the S586A variant displayed increased catalytic efficiency and substrate affinity, which is attributed to decreased steric hindrance and increased hydrophobicity, as verified by the saturation mutagenesis at S586. Our results provide structural information about the residues important for the protein engineering of TDC to improve catalytic efficiency in the green manufacturing of tyramine.