Origins of Enantiopreference of Mycobacterium smegmatis Acyl Transferase: A Computational Analysis

Abstract

Acyl transferase from Mycobacterium smegmatis (MsAcT) is a promising biocatalyst because it catalyzes an acyl transfer reaction in aqueous solution, thereby accepting many primary and secondary alcohols as substrates. MsAcT also exhibits high enantioselectivity for a selected number of secondary alcohols. To increase the applicability of this enzyme for the production of optically active compounds, a detailed understanding of the reaction mechanism and the factors that affect enantioselectivity is essential. Herein, quantum chemical calculations are employed to study the reactions of two secondary alcohols, 1‐isopropyl propargyl alcohol and 2‐hydroxy propanenitrile, for which the enzyme displays opposite enantiopreference, favoring the S enantiomer in the former case and R enantiomer in the latter. A model of the active site has been designed and for both substrates various binding modes are evaluated and the intermediates and transition states along the reaction path are then located. The calculated energy profiles agree with the experimental observations, and reproduce the selectivity outcome. Through a detailed analysis of the geometries of key transition states, insights into the origins of the enantiopreference are obtained.

Protein Engineering for Enhanced Acyltransferase Activity, Substrate Scope, and Selectivity of the Mycobacterium smegmatis Acyltransferase MsAcT

“…15,36 Additionally, sites suggested by computational studies to be relevant to acyltransferase activity were investigated. 35,37 Binding Site and Tunnel Analyses. Residues forming the active site of MsAcT (PDB code: 2Q0Q) were examined.…”

“…Another recently published computational study suggested that W16, A55, F150, and F154 are important in determining the enantioselectivity of MsAcT. 37 This hypothesis was based on differences in the stabilizing effects these residues have on transition states, depending on the substrate enantiomer used. Because of their roles in stabilization of transition states, these residues probably also influence acyl transfer efficiency and are therefore potential hotspots for improving the AT:H ratio and enantioselectivity of MsAcT.…”

“…In order to identify target residues for site-directed mutagenesis, the crystal structures of MsAcT with and without a covalently bound inhibitor (PDB codes: 2Q0S and 2Q0Q, respectively) were inspected and compared to the homologous Est24 from Sinorhizobium meliloti (PDB code: 5HOE). , Additionally, sites suggested by computational studies to be relevant to acyltransferase activity were investigated. , …”

The Unexplored Importance of Fleeting Chiral Intermediates in Enzyme-Catalyzed Reactions

“…The extensive literature of esterases reveals many important mechanistic details, but no information on the chirality of the respective oxyanions is explicitly discussed. 14,18,113,114 The literature on the mechanism of proteases is even more vast, and the chirality of oxyanions has also been routinely ignored. Here, we mention only a single example case.…”

“…The extensive literature of esterases reveals many important mechanistic details, but no information on the chirality of the respective oxyanions is explicitly discussed. ,,, …”