Certain hydrolases preferentially catalyze acyl transfer over hydrolysis in an aqueous environment. However, the molecular and structural reasons for this phenomenon are still unclear.H erein, we provide evidence that acyltransferase activity in esterases highly correlates with the hydrophobicity of the substrate-binding pocket. Ah ydrophobicity 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,l eading to the discovery of five novel promiscuous acyltransferases.W ea lso developed as imple yet versatile colorimetric assayfor rapid characterization of novel acyltransferases.T his study demonstrates that promiscuous acyltransferase activity is not as rare as previously thought and provides access to avast number of novel acyltransferases with diverse substrate specificity and potential applications.

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 activity-based 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 4-chlorobenzoate and 4-(2-aminoethyl)morpholine (ca. 20 % conversion). We solved the crystal structure of EstCE1 and detailed structure-function analysis revealed a three-amino acid motif important for promiscuous acyltransferase activity. Introducing this motif into an esterase without acetyltransferase activity transformed a "hydrolase" into an "acyltransferase".

The highly efficient and versatile acyltransferase MsAcT from Mycobacterium smegmatis catalyzes aqueous acyl transfer reactions, enabling applications in environmentally friendly processes and enzyme cascades. We rationally designed several variants with up to 30-fold increased acyl transfer to hydrolysis ratios while mostly retaining initial activity. Variants exhibiting broader acyl-donor substrate scope and higher or inverted enantioselectivity were also designed. Alterations of the catalytic His-Asp pair decreased the activation of hydrolytic water, thereby increasing acyl transfer to hydrolysis ratios. This study demonstrates that targeting the disruption of water networks and manipulating the activation of nucleophiles are promising strategies for engineering promiscuous acyltransferase activities.

Promiscuous hydrolases/acyltransferases have attracted attention for their ability to efficiently catalyze selective transacylation reactions in water to produce esters, thioesters, amides, carbonates, and carbamates. Promiscuous hydrolases/acyltransferases can be implemented into aqueous enzyme cascades and are ideal biocatalysts for the acylation of hydrophilic substrates that are barely soluble in dry organic solvents. This activity was thought to be rare, and recent research has focused on just a small number of accidentally identified promiscuous hydrolases/acyltransferases. High-throughput screening for acyltransferases and an in silico sequence-based method for prediction of acyltransferase activity provided access to many efficient promiscuous hydrolases/acyltransferases, thereby demonstrating that promiscuous acyltransferase activity is rather common in hydrolases. These synthetically valuable enzymes could further be enhanced by protein engineering. This Perspective aims to demonstrate the synthetic potential of these enzymes and raise awareness of the frequency of this activity.

Acyltransferases are enzymes that are capable of catalyzing the transesterification of non-activated esters in an aqueous environment and therefore represent interesting catalysts for applications in various fields. However, only a few acyltransferases have been identified so far, which can be explained by the lack of a simple, broadly applicable high-throughput assay for the identification of these enzymes from large libraries. Here, we present the development of such an assay that is based on the enzymatic formation of oligocarbonates from dimethyl carbonate and 1,6-hexanediol. In contrast to the monomers used as substrates, the oligomers are not soluble in the aqueous environment and form a precipitate which is used to detect enzyme activity by the naked eye, by absorbance or by fluorescence measurements. With activity detected and thus confirmed for the enzymes Est8 and MsAcT, the assay enabled the first identification of acyltransferases that act on carbonates. It will thus allow for the discovery of further efficient acyltransferases or of more efficient variants via enzyme engineering.