Wu Jingyue, Crotti Michele, Bassanini Ivan, Hassankalhori Mahdi, Ferrandi Erica Elisa, Sancho Ferran, Monti Daniela, Castagnolo Daniele
Department of Chemistry, University College London, Marshgate Building, Manufacturing Futures Lab, 7 Sidings Street, London, E20 2AE, UK.
Istituto di Scienze e Tecnologie Chimiche "Giulio Natta", Consiglio Nazionale delle Ricerche, Via Mario Bianco 9, Milano, 20131, Italy.
Angew Chem Int Ed Engl. 2025 Jul;64(29):e202505032. doi: 10.1002/anie.202505032. Epub 2025 May 29.
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.
内酯酶是一类具有催化多效性的金属酶，已从生物学角度进行了广泛研究，但其作为生物催化剂的应用仍未得到充分探索。在本研究中，我们揭示了内酯酶在手性C3-取代-γ-硫代内酯的水解和消旋化以及γ-硫代-α-取代羧酸的不对称合成中的生物催化活性。研究了来自不同蛋白质超家族的内酯酶的硫代内酯酶活性。来自金属β-内酰胺酶样内酯酶家族的生物催化剂GcL催化了同型半胱氨酸（Hcy）硫代内酯的酶促动力学拆分（EKR），具有优异的对映选择性（E值高达136），产生了对映体富集的Hcy硫代内酯和高对映体过量的γ-硫代-α-氨基羧酸。此外，生物催化剂N9 Y71G是重组祖先对氧磷酶N9的合理工程变体，催化了C3-硫代-γ-硫代内酯的动态动力学拆分（DKR），以对映选择性方式产生了高对映体过量（高达>99%）和高收率（高达>99%）的γ-硫代-α-硫代羧酸。通过计算和定点诱变研究获得了关于内酯酶生物催化剂的作用机制和立体选择性的见解。
