Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution

Abstract

Engineering artificial enzymes with high activity and catalytic mechanism different from naturally occurring enzymes is a challenge in protein design. For example, many attempts have been made to obtain active hydrolases by introducing a Ser → Cys exchange at the respective catalytic triads, but this generally induced a breakdown of activity. We now report that this long-standing dogma no longer pertains, provided additional mutations are introduced by directed evolution. By employing Candida antarctica lipase B (CALB) as the model enzyme with the Ser-His-Asp catalytic triad, a highly active cysteine-lipase having a Cys-His-Asp catalytic triad and additional mutations W104V/A281Y/A282Y/V149G can be evolved, showing a 40-fold higher catalytic efficiency than wild-type CALB in the hydrolysis of 4-nitrophenyl benzoate, and tolerating bulky substrates. Crystal structures, kinetics, MD simulations and QM/MM calculations reveal dynamic features and explain all results, including the preference of a two-step mechanism involving the zwitterionic pair Cys105 − /His224 + rather than a concerted process.

Enzymatic Synthesis of Poly(caprolactone): A QM/MM Study

“…Although this reported value can be used as reference, we cannot compare it directly with the free enzyme, since enzyme immobilization can negatively affect the activity of the enzyme due to diffusion restrictions . Nevertheless, our calculated value of 9.8 kcal/mol for ring‐opening by the free form of CalB is in good agreement with previous studies that point to values around 10.0–11.0 kcal/mol . The orientation of the histidine/aspartate residues in the INT‐1 and connected transition states ( TS 1 and TS 2 ) in the 1D, 2D‐PMFs and in the small cluster models (Figure S1, Figure B, Figure B and Figure ) offer an explanation for the enzymes energy differences.…”

“…The nucleophilic attack proceeds via the formation of a first transition state structure ( TS 1 ), which has a free energy barrier (ΔG ≠ ) of 8.8±0.1 kcal/mol (Figure A) generating the INT‐1 (Figure B). As previously reported, the TS 1 and all other transition states in this catalytic cycle are concerted, meaning that bond making/breaking events occur simultaneously with a proton transfer to or from the histidine. In INT‐1 the backbone amide groups of the oxyanion hole Q106 and T40 stabilize the developing negative charge on the substrate oxygen atom (O oxy ) (1.90±0.12 Å and 2.24±0.32 Å, respectively, Figure B).…”

Scalable molecular dynamics on CPU and GPU architectures with NAMD

“…192 For most applications, hybrid QM/MM protocols are wellestablished and have been employed broadly to investigate a variety of biomolecular problems, most notably in enzymatic activity, including the mechanism of HIV integrases, glutamine synthetases, glycoside hydrolases, dioxygenases, lipases, dehydrogenases, catalases, glycosyltransferases, and nearly every other class of enzymes with known structure. [193][194][195][196][197][198][199] The large list of enzymatic mechanisms that have been explored showcases the significant contribution of QM/MM methods to the development of enzymology 200 . In addition, polarization effects have also been the focus of QM/MM simulations.…”

Coupling biocatalysis with high-energy flow reactions for the synthesis of carbamates and β-amino acid derivatives

“…The possibility to exploit the use of an enzyme to derivatize benzyl alcohol into an easily separable species was an intriguing opportunity. To integrate such a biocatalyzed transformation into the continuous Curtius rearrangement process, immobilized CALB, a robust hydrolase enzyme was utilized as it is frequently used for esterification reactions [ 26 – 28 ]. CALB could convert benzyl alcohol in the presence of vinyl butyrate into benzyl butyrate and acetaldehyde.…”