Fluorogenic structure activity library pinpoints molecular variations in substrate specificity of structurally homologous esterases

Abstract

Cellular esterases catalyze many essential biological functions by performing hydrolysis reactions on diverse substrates. The promiscuity of esterases complicates assignment of their substrate preferences and biological functions. To identify universal factors controlling esterase substrate recognition, we designed a 32-member structure-activity relationship (SAR) library of fluorogenic ester substrates and used this library to systematically interrogate esterase preference for chain length, branching patterns, and polarity to differentiate common classes of esterase substrates. Two structurally homologous bacterial esterases were screened against this library, refining their previously broad overlapping substrate specificity. esterase ybfF displayed a preference for γ-position thioethers and ethers, whereas Rv0045c from displayed a preference for branched substrates with and without thioethers. We determined that this substrate differentiation was partially controlled by individual substrate selectivity residues Tyr-119 in ybfF and His-187 in Rv0045c; reciprocal substitution of these residues shifted each esterase's substrate preference. This work demonstrates that the selectivity of esterases is tuned based on transition state stabilization, identifies thioethers as an underutilized functional group for esterase substrates, and provides a rapid method for differentiating structural isozymes. This SAR library could have multifaceted future applications, including imaging, biocatalyst screening, molecular fingerprinting, and inhibitor design.

Structure-guided microbial targeting of antistaphylococcal prodrugs

“…To facilitate design of microbially targeted prodrug activation using these two enzymes, we next sought to extensively characterize GloB and FrmB substrate specificity. We employed a 32-compound ester substrate library, which fluoresces upon esterase activity (Supplementary Figure 5) 36 . This library systematically varies ester substrate length, branching patterns, and ether and sulfide positioning, thereby allowing for the precise determination of structure-activity relationships.…”

“…POM-HEX, Hemi-HEX, and HEX were synthesized and resuspended in 100% DMSO as described previously 12 . Fluorescent ester compounds were generously provided by the laboratory of Geoffrey Hoops (Butler University), and synthesis and characterization has been previously described 36 . Pooled, delipidated, defibrinated, and lyophilized human and mouse serum was obtained from Rockland Inc.…”

From Differential Stains to Next Generation Physiology: Chemical Probes to Visualize Bacterial Cell Structure and Physiology

“…A multitude of different fluorophores/fluorescent tags with different optical and physico-chemical properties are available that cater to the demands of multiplexed imaging or advanced detection methods (e.g., super-resolution imaging or in vivo imaging). Furthermore, fluorogenic probes [10][11][12][13] and quenched fluorescent probes [10,14] are activatable probes with reduced background fluorescence signals and higher specificity compared to targeted fluorescent probes, making them particularly useful for real time imaging studies. Despite this versatility, the physico-chemical properties of fluorescent tags can dramatically alter the biological activity and distribution (e.g., cellular uptake) of the biomolecule of interest.…”

“…Commercially available fluorogenic substrates are most commonly based on 7-amino-4 carbamoylmethylcoumarin (ACC) or p-nitroaniline (pNA) fluorogens. A number of alternative fluorogenic reporters are emerging, such as acyloxymethyl ether fluorescein [13] or 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO)-derivatives, which emit in the far-red spectrum [30,31], or spontaneously-blinking fluorogens for super-resolution microscopy [32].…”

“…In addition to a masked fluorophore or luminophore, a substrate probe must contain a specific chemical group (e.g., an ester or peptide bond) that is actively modified by enzyme targets (e.g., an esterase or protease) and that is part of a larger recognition element that may discriminate between different members of the same enzyme family. These recognition elements include e.g., peptide sequences that are processed differently based on the preferences of proteases with different substrate specificity profiles [12], different acyl groups in ester substrates [7,13,30,31,85,[100][101][102] and different sugar moieties in glycosidases substrates. One example of the molecular diversity that such probes can cover is a 32-member library of fluorogenic esterase substrates that Johnson and coworkers developed for structure-activity relationship studies [13].…”