Crystal structures of a novel family IV esterase in free and substrate‐bound form

Abstract

Bacterial lipolytic enzymes of family IV are homologs of the mammalian hormone‐sensitive lipases (HSL) and have been successfully used for various biotechnological applications. The broad substrate specificity and ability for enantio‐, regio‐, and stereoselective hydrolysis are remarkable features of enzymes from this class. Many crystal structures are available for esterases and lipases, but structures of enzyme–substrate or enzyme–inhibitor complexes are less frequent although important to understand the molecular basis of enzyme–substrate interaction and to rationalize biochemical enzyme characteristics. Here, we report on the structures of a novel family IV esterase isolated from a metagenomic screen, which shows a broad substrate specificity. We solved the crystal structures in the apo form and with a bound substrate analogue at 1.35 and 1.81 Å resolution, respectively. This enzyme named PtEst1 hydrolyzed more than 60 out 96 structurally different ester substrates thus being substrate promiscuous. Its broad substrate specificity is in accord with a large active site cavity, which is covered by an α‐helical cap domain. The substrate analogue methyl 4‐methylumbelliferyl hexylphosphonate was rapidly hydrolyzed by the enzyme leading to a complete inactivation caused by covalent binding of phosphinic acid to the catalytic serine. Interestingly, the alcohol leaving group 4‐methylumbelliferone was found remaining in the active site cavity, and additionally, a complete inhibitor molecule was found at the cap domain next to the entrance of the substrate tunnel. This unique situation allowed gaining valuable insights into the role of the cap domain for enzyme–substrate interaction of esterases belonging to family IV.DatabaseStructural data of PtEst1 are available in the worldwide protein data bank (https://www.rcsb.org) under the accession codes: 6Z68 (apo‐PtEst1) and 6Z69 (PtEst1‐inhibitor complex).

A Plurizyme with Transaminase and Hydrolase Activity Catalyzes Cascade Reactions

“…Briefly, the process begins with scanning the transaminase surface to identify noncatalytic ester binding sites using global Protein Energy Landscape Exploration (PELE) exploration [31] . Next, we perform local explorations of active site variants to introduce a well‐positioned catalytic triad while also considering the existence of oxyanion holes that are required for efficient ester hydrolysis [32] . Finally, optimum variants designed with PELE are ranked through i) Molecular Dynamics (MD) refinement simulations, accounting for the integrity of the triad, and ii) ΔΔ G estimations using the HotSpot Wizard.…”

Crystal structure of a family VIII β‐lactamase fold hydrolase reveals the molecular mechanism for its broad substrate scope

“…In addition, sequence‐substrate spectrum relationships have revealed that substrate promiscuity is a characteristic most prevalent in members of family IV, also called the hormone‐sensitive lipase (HSL) family [ 4 ]. Recent structural analysis has suggested that the substrate promiscuity that was frequently observed for this esterase family is due to a substrate prerecognition mechanism by hydrophobic patches of the cap domains [ 6 ].…”

Molecular Characterization of Novel Family IV and VIII Esterases from a Compost Metagenomic Library

“…The bHSL is divided into two domains: cap and catalytic domains. The catalytic domain forms α-helix and β-sheets with catalytic residues, and the cap domain is located on the upper side of the catalytic residues, which is associated with substrate recognition [ 8 ].…”

“…The DNA sample is directly extracted from sediments [ 13 ] and is called environmental DNA (eDNA) [ 14 , 15 ], serving extremely diverse genes. Numerous recent studies have focused on metagenomes from various environments, such as Antarctic soil [ 16 ], deep-sea [ 17 ], oil-polluted mudflat [ 18 ], volcano soil [ 19 ], and plant wasted water [ 8 ].…”