Unlocking Lactonase Enzymes as Biocatalysts for the Deracemisation of Chiral γ-Thiolactones

Abstract

Lactonases, a class of metalloenzymes that exhibit catalytic promiscuity, have been extensively studied from a biological perspective, yet their application as biocatalysts remains underexplored. In this study, we disclose the biocatalytic activity of lactonase enzymes in the hydrolysis and deracemisation of chiral C3-substituted-γ-thiolactones and the asymmetric synthesis of γ-thio-α-substituted-carboxylic acids. The thiolactonase activity of lactonases from different protein superfamilies was investigated. The biocatalyst GcL, from the metallo-β-lactamase-like lactonase family, catalysed the enzymatic kinetic resolution (EKR) of homocysteine (Hcy) thiolactones with excellent enantioselectivity (E-value up to 136), yielding enantioenriched Hcy thiolactones and γ-thio-α-amino-carboxylic acids with high ees. Additionally, the biocatalyst N9 Y71G, a rationally engineered variant of the reconstructed ancestral paraoxonase enzyme N9, catalysed the dynamic kinetic resolution (DKR) of C3-thio-γ-thiolactones, yielding γ-thio-α-thio-carboxylic acids in enantioselective manner with high ees (up to >99%) and yields (up to >99%). Insights on the mechanism and the stereoselectivity of the lactonase biocatalysts were gained through computational and site-directed mutagenesis studies.

Keywords: Biocatalysis; Kinetic resolution; Lactonase; Sulphur compounds; Thiolactones.

Figure 1

Previous works on lactonase biocatalysts and aims of the present work.

Figure 2

a) Docking of (R)‐1a (yellow) and (S)‐1a (green) enantiomers in the GcL catalytic site and interaction of (S)‐1a with metal cofactors and amino acid residues; b) proposed mechanism for the GcL biocatalysed EKR of thiolactones 1.^{[ 16} , ^{79 ]} Additional information on docking analysis is provided in Table S10.

Figure 3

a) Catalytic pocket of N9. b) Docking of thiolactone (R)‐5a (pink) into N9 catalytic pocket showing the steric hindrance of the residue Tyr₇₁ (Y71). The surface area of Tyr₇₁ is highlighted in green. c) Docking of thiolactone (R)‐5a (pink) into N9 Y71A mutant catalytic pocket. d) Docking of thiolactone (R)‐5a (pink) into N9 Y71G mutant catalytic pocket.

Scheme 1

Mechanism of the DKR of 3‐thiosubstituted thiolactones 5.

Scheme 2

a) Racemisation experiment of enantioenriched thiolactone (R)‐5a in Tris‐HCl buffer, pH 9.0. b) Racemisation experiments of enantioenriched acid (R)‐6a in Tris‐HCl buffer, pH 9.0. c) Deuterium labelling experiment on thiolactone (R)‐5a. ¹H‐NMR spectra of the thiolactone (R)‐5a before (above) and after (below) suspension in deuterated buffer.

Scheme 3

a) Biocatalytic hydrolysis of lactone 8a with N9 and N9 Y71G lactonases. b) Biocatalytic hydrolysis of lactone 8b with N9 lactonase. c) Biocatalytic hydrolysis of 3‐phenoxy‐thiolactone 10 with N9 and N9 Y71G lactonases.

Scheme 4

Lactonisation of (R)‐9b and configurational stability experiments.

Figure 4

a) Coordination of the carbonyl moiety of (R)‐5a with His₁₄₄ and coordination of His₁₄₄ with His₁₃₃. b) Amino acid residues of the N9 Y71G catalytic pocket interacting with (R)‐5a. c) Proposed mechanism of action of N9 Y71G.

Figure 5

Plotted clusters of (R)‐5a and (S)‐5a docking results into N9 and N9 Y71G modelled structures. a) and b) in both N9 and N9 Y71G, there are docking pose clusters of (R)‐5a falling into the dash‐lined box that highlights the clusters that satisfy the affinity score and distance criteria, based on the catalytic orientation of the substrate in the vicinity of the catalytic Ca²⁺ ion. c) and d) no clusters of (S)‐5a docked into N9 or N9 Y71G fell into the selected box; however, some low‐scoring poses (indicated by dashed circle) with the ideal O–Ca distance and orientation were seen in N9.