Engineering of Halide Methyltransferase BxHMT through Dynamic Cross-Correlation Network Analysis.

Halide methyltransferases (HMTs) provide an effective way to regenerate S-adenosyl methionine (SAM) from S-adenosyl homocysteine and reactive electrophiles, such as methyl iodide (MeI) and methyl toluene sulfonate (MeOTs). As compared with MeI, the cost-effective unnatural substrate MeOTs can be accessed directly from cheap and abundant alcohols, but shows only limited reactivity in SAM production. In this study, we developed a dynamic cross-correlation network analysis (DCCNA) strategy for quickly identifying hot spots influencing the catalytic efficiency of the enzyme, and applied it to the evolution of HMT from Paraburkholderia xenovorans. Finally, the optimal mutant, M4 (V55T/C125S/L127T/L129P), exhibited remarkable improvement, with a specific activity of 4.08 U/mg towards MeOTs, representing an 82-fold increase as compared to the wild-type (WT) enzyme. Notably, M4 also demonstrated a positive impact on the catalytic ability with other methyl donors. The structural mechanism behind the enhanced enzyme activity was uncovered by molecular dynamics simulations. Our work not only contributes a promising biocatalyst for the regeneration of SAM, but also offers a strategy for efficient enzyme engineering.

Engineering a norcoclaurine synthase for one-step synthesis of (S)-1-aryl-tetrahydroisoquinolines.

Tetrahydroisoquinoline alkaloids (THIQAs) are ubiquitous compounds with important pharmaceutical and biological activity. Their key N-heterocyclic structural motifs are synthesised via Pictet-Spengler (P-S) reaction by norcoclaurine synthases (NCS) in plants. The synthesis of 1-aryl-tetrahydroisoquinoline alkaloids has attracted increasing attention due to their antitumor and antivirus activities. Herein, the L68T/M97V mutant of NCS from Thalictrum flavum with improved activity was developed by semi-rational design. This mutant not only showed higher catalytic performance (> 96% conversion) toward benzaldehyde and dopamine over the wild-type enzyme, but also catalysed the P-S reaction of the bulky substrate 4-biphenylaldehyde and dopamine with high conversion (> 99%) for the effective synthesis of 1-aryl-THIQA. In terms of stereoselectivity, all products synthesised by the L68T/M97V mutant showed high optical purity (92-99% enantiomeric excess).

Target-oriented discovery of a new esterase-producing strain Enterobacter sp. ECU1107 for whole cell-catalyzed production of (2S,3R)-3-phenylglycidate as a chiral synthon of Taxol.

A new strain, Enterobacter sp. ECU1107, was identified among over 200 soil isolates using a two-step screening strategy for the enantioselective synthesis of (2S,3R)-3-phenylglycidate methyl ester (PGM), a key intermediate for production of a potent anticancer drug Taxol®. An organic-aqueous biphasic system was employed to reduce spontaneous hydrolysis of the substrate PGM and isooctane was found to be the most suitable organic solvent. The temperature and pH optima of the whole cell-mediated bioreaction were 40 °C and 6.0, respectively. Under these reaction conditions, the enantiomeric excess (ee(s)) of (2S,3R)-PGM recovered was greater than 99 % at approximately 50 % conversion. The total substrate loading in batch reaction could reach 600 mM. By using whole cells of Enterobacter sp. ECU1107, (2S,3R)-PGM was successfully prepared in decagram scale in a 1.0-l mechanically stirred reactor, affording the chiral epoxy ester in >99 % ee s and 43.5 % molar yield based on the initial load of racemic substrate.

Efficient production of (R)-o-chloromandelic acid by deracemization of o-chloromandelonitrile with a new nitrilase mined from Labrenzia aggregata.

(R)-o-Chloromandelic acid is the key precursor for the synthesis of Clopidogrel®, a best-selling cardiovascular drug. Although nitrilases are often used as an efficient tool in the production of α-hydroxy acids, there is no practical nitrilase specifically developed for (R)-o-chloromandelic acid. In this work, a new nitrilase from Labrenzia aggregata (LaN) was discovered for the first time by genomic data mining, which hydrolyzed o-chloromandelonitrile with high enantioselectivity, yielding (R)-o-chloromandelic acid in 96.5% ee. The LaN was overexpressed in Escherichia coli BL21 (DE3), purified, and its catalytic properties were studied. When o-chloromandelonitrile was used as the substrate, the V(max) and K(m) of LaN were 2.53 µmol min⁻¹ mg⁻¹ protein and 0.39 mM, respectively, indicating its high catalytic efficiency. In addition, a study of substrate spectrum showed that LaN prefers to hydrolyze arylacetonitriles. To relieve the substrate inhibition and to improve the productivity of LaN, a biphasic system of toluene-water (1:9, v/v) was adopted, in which o-chloromandelonitrile of 300 mM (apparent concentration, based on total volume) could be transformed by LaN in 8 h, giving an isolated yield of 94.5%. The development of LaN makes it possible to produce (R)-o-chloromandelic acid by deracemizing o-chloromandelonitrile with good ee value and high substrate concentration.