Sequence‐Based 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 substrate‐binding 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.

New insights into the function of plant tannase with promiscuous acyltransferase activity

“…Sphingolipid ceramide N ‐deacylase from Pseudomonas and acyl transferase (MsAcT) with an unusual architecture from Mycobacterium smegmatis can catalyze acyl transfer reactions (Kita et al., 2001; Mathews et al., 2007). As a unique property, the acyl transfer function of some hydrolases has received extensive attention and has been applied in biocatalysis in biotransformation (Bruggink et al., 1998; Kasche, 1986; Müller et al., 2015; Müller, Becker, et al., 2020; Müller, Godehard, et al., 2020). For example, Mayer et al.…”

Promiscuous Dehalogenase Activity of the Epoxide Hydrolase CorEH from Corynebacterium sp. C12

“…12,13 Some dehalogenases, DmxA, 14 DsaA, 15 DmrB, 16 Within protein superfamilies the primary function of one family member is often found as promiscuous activity in other family members and the same promiscuous activity is usually shared by more than one family member. 18,19 Rather than rare exceptions, promiscuous activities are frequently observed, and it is commonly believed that catalytic promiscuity plays an important role in the emergence of novel functions by providing a starting point for evolution by natural selection. 18 The versatility of the catalytic triad employed by α/βhydrolases allows for the catalysis of several distinct chemical reactions, explaining why catalytic promiscuity is often observed.…”

Efficient Acylation of Sugars and Oligosaccharides in Aqueous Environment Using Engineered Acyltransferases

“…45 technique, we identified several promiscuous acyltransferases in the bacterial hormone-sensitive lipase (bHSL) family. 46 Product formation catalyzed by promiscuous acyltransferases is kinetically controlled, which means that a maximum product concentration is rapidly reached before the product is hydrolyzed due to the thermodynamic equilibrium. 46,47 It is believed that hydrophobicity in the active site of promiscuous acyltransferases and high affinity toward the acyl acceptor are key requirements for facilitating binding of the acyl acceptor for acyl transfer and preventing water from hydrolyzing the acyl-enzyme intermediate.…”

“…46 Product formation catalyzed by promiscuous acyltransferases is kinetically controlled, which means that a maximum product concentration is rapidly reached before the product is hydrolyzed due to the thermodynamic equilibrium. 46,47 It is believed that hydrophobicity in the active site of promiscuous acyltransferases and high affinity toward the acyl acceptor are key requirements for facilitating binding of the acyl acceptor for acyl transfer and preventing water from hydrolyzing the acyl-enzyme intermediate. 46,48,49 Unfortunately, their highly hydrophobic active sites 46 (Figure S1) so far mostly enable acyl transfer to hydrophobic, aromatic substrates and limit their applicability for sugar acylation in aqueous reaction systems.…”