15 research outputs found
Advanced database mining integrating sequence and structure bioinformatics with microfluidics challenges enzyme engineering
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Enzyme Kits to Facilitate the Integration of Biocatalysis into Organic Chemistry â First Aid for Synthetic Chemists
Abstract
First Aid Kits are collections of the most important medical equipment required for quick medical assistance. Similarly, enzyme kits can provide a proficient, readyâ and easyâtoâuse collection of biocatalysts that can be applied with high reproducibility. In this article, we illustrate how kits of oxyfunctionalisation enzymes could operate as synthetic âFirst Aidâ for chemists working on complex natural product total synthesis in an earlyâ or lateâstage fashion, as well as in lead diversification in drug discovery processes. We reason that enzyme kits could catalyse the integration of biocatalysis into (synthetic) organic chemistry and describe how we envision their future application
From Natural Methylation to Versatile Alkylations Using Halide Methyltransferases
Abstract
Halide methyltransferases (HMTs) enable the enzymatic synthesis of Sâadenosylâlâmethionine (SAM) from Sâadenosylâlâhomocysteine (SAH) and methyl iodide. Characterisation of a range of naturally occurring HMTs and subsequent protein engineering led to HMT variants capable of synthesising ethyl, propyl, and allyl analogues of SAM. Notably, HMTs do not depend on chemical synthesis of methionine analogues, as required by methionine adenosyltransferases (MATs). However, at the moment MATs have a much broader substrate scope than the HMTs. Herein we provide an overview of the discovery and engineering of promiscuous HMTs and how these strategies will pave the way towards a toolbox of HMT variants for versatile chemoâ and regioselective biocatalytic alkylations
Enzymatic Characterization and Elucidation of the Catalytic Mechanism of a Recombinant Bovine Glycine N
Entdeckung und Design promiskuitiver AcyltransferaseâAktivitĂ€t in Carboxylesterasen der Familie VIII
Entdeckung und Design promiskuitiver AcyltransferaseâAktivitĂ€t in Carboxylesterasen der Familieâ VIII
Discovery and Design of Familyâ VIII Carboxylesterases as Highly Efficient Acyltransferases
Abstract
Promiscuous acyltransferase activity is the ability of certain hydrolases to preferentially catalyze acyl transfer over hydrolysis, even in bulk water. However, poor enantioselectivity, low transfer efficiency, significant product hydrolysis, and limited substrate scope represent considerable drawbacks for their application. By activityâbased screening of several hydrolases, we identified the family VIII carboxylesterase, EstCE1, as an unprecedentedly efficient acyltransferase. EstCE1 catalyzes the irreversible amidation and carbamoylation of amines in water, which enabled the synthesis of the drug moclobemide from methyl 4âchlorobenzoate and 4â(2âaminoethyl)morpholine (ca. 20â% conversion). We solved the crystal structure of EstCE1 and detailed structureâfunction analysis revealed a threeâamino acid motif important for promiscuous acyltransferase activity. Introducing this motif into an esterase without acetyltransferase activity transformed a âhydrolaseâ into an âacyltransferaseâ
SequenceâBased Prediction of Promiscuous Acyltransferase Activity in Hydrolases
Abstract
Certain hydrolases preferentially catalyze acyl transfer over hydrolysis in an aqueous environment. However, the molecular and structural reasons for this phenomenon are still unclear. Herein, we provide evidence that acyltransferase activity in esterases highly correlates with the hydrophobicity of the substrateâbinding pocket. A hydrophobicity scoring system developed in this work allows accurate prediction of promiscuous acyltransferase activity solely from the amino acid sequence of the cap domain. This concept was experimentally verified by systematic investigation of several homologous esterases, leading to the discovery of five novel promiscuous acyltransferases. We also developed a simple yet versatile colorimetric assay for rapid characterization of novel acyltransferases. This study demonstrates that promiscuous acyltransferase activity is not as rare as previously thought and provides access to a vast number of novel acyltransferases with diverse substrate specificity and potential applications