Dramatically improved catalytic activity of an artificial (S)-selective arylmalonate decarboxylase by structure-guided directed evolution

Abstract

Using three rounds of structure-guided directed evolution, the catalytic activity of the (S)-selective arylmalonate decarboxylase variant G74C/C188S could be increased up to 920-fold. The best variant had a 220-fold improved activity in the production of (S)-naproxen with excellent enantioselectivity (>99% ee).

Engineered hydrophobic pocket of (S)-selective arylmalonate decarboxylase variant by simultaneous saturation mutagenesis to improve catalytic performance

“…The decarboxylation activity toward 1a in these cell-free extracts was screened by a highthroughput pH-dependent color assay. 14,16) In the activity screening for crude extracts, variants G74C/V156I/ M159L/C188G (CLG-I) and G74C/V156L/M159L/ C188G (CLG-L) both displayed higher activity compared with the template CLG ( Table 2). After the variants were purified, their activities were quantitatively investigated based on the HPLC-detection of 2a.…”

“…As a starting variant, we chose G74C/M159L/C188G (CLG), which thus far shows the highest activity toward 1a among the (S)-selective AMDase variants. 16) L40, V156, and M73 of CLG were mutated simultaneously using the degenerate codon VTK that donates Leu, Ile, Val, and Met. To ensure 95% coverage of the mutant gene library coding L40X/M73X/V156X (X = L, I, V and M), more than 646 transformants are required.…”

“…The V43I/G74C/A125P/V156L/M159L/C188G mutant enzyme (CLGL-IP) demonstrated a 9,500-fold improvement toward 1a compared with the weakly active (S)-selective AMDase variant G74C/C188S. Therefore, throughout our directed evolution studies in the previous 16) and present research, the hydrophobic pocket has been optimized into a more suitable form to drive the decarboxylation of α-methylated arylmalonates. Furthermore, the notable result showing that CLGL-IP demonstrated higher activity toward 1c than the (R)-selective wild-type AMDase indicates the possibility that the improved AMDase variants could be applied for the synthesis of (S)-ibuprofen.…”

“…A synergistic effect on enzymatic activity would be expected following multiple mutations in the active site, 19,20) but this is difficult to obtain via the iterative site-directed saturation mutagenesis performed in previous studies. 14,16) …”

“…16) Next, we analyzed the substrate specificity of the variants. G74C/M159L/ C188G showed the highest specific activity toward α-aryl-α-methylmalonates (1a-c).…”