Over 100 metabolic serine hydrolases are present in humans with confirmed functions in metabolism, immune response, and neurotransmission. Among potentially clinically-relevant but uncharacterized human serine hydrolases is OVCA2, a serine hydrolase that has been linked with a variety of cancer-related processes. Herein, we developed a heterologous expression system for OVCA2 and determined the comprehensive substrate specificity of OVCA2 against two ester substrate libraries. Based on this analysis, OVCA2 was confirmed as a serine hydrolase with a strong preference for long-chain alkyl ester substrates (>10-carbons) and high selectivity against a variety of short, branched, and substituted esters. Substitutional analysis was used to identify the catalytic residues of OVCA2 with a Ser117-His206-Asp179 classic catalytic triad. Comparison of the substrate specificity of OVCA2 to the model homologue FSH1 from Saccharomyces cerevisiae illustrated the tighter substrate selectivity of OVCA2, but their overlapping substrate preference for extended straight-chain alkyl esters. Conformation of the overlapping biochemical properties of OVCA2 and FSH1 was used to model structural information about OVCA2. Together our analysis provides detailed substrate specificity information about a previously, uncharacterized human serine hydrolase and begins to define the biological properties of OVCA2. OPEN ACCESS Citation: Bun JS, Slack MD, Schemenauer DE, Johnson RJ (2020) Comparative analysis of the human serine hydrolase OVCA2 to the model serine hydrolase homolog FSH1 from S. cerevisiae. PLoS ONE 15(3): e0230166. https://doi.org/ 10.

Metabolic G-protein Coupled Receptors (GPCRs). (A) Intramembrane access to the binding pocket of GPR40 (also known as free fatty acid receptor 1; PDB code: 4PHU). The binding pocket of GPR40 (grey) is covered by extracellular loop 2 (ECL2; cyan) preventing entry from the extracellular space. Instead the allosteric regulator, TAK-875 (pink), accesses the binding pocket through the plasma membrane. (B) Structural determination of the lysophosphatidic acid receptor (LPA 1 ; PDB code: 4Z34). LPA 1 was crystallized with a stabilizing Cytochrome b 562 RIL subunit (circled in orange) inserted into the third intracellular loop and with membrane lipids bound to help orient LPA 1 in the plasma membrane. (C) Pharmacological regulation of metabotropic glutamate receptor 5 (mGlu5; PDB code: 4OO9). Slab view of the allosteric binding site (allosteric regulator mavoglurant (red)) within the 7-transmembrane helices of mGlu5 (green).

Mycobacterium tuberculosis has a complex life cycle transitioning between active and dormant growth states depending on environmental conditions. LipN (Rv2970c) is a conserved mycobacterial serine hydrolase with regulated catalytic activity at the interface between active and dormant growth conditions. LipN also catalyzes the xenobiotic degradation of a tertiary ester substrate and contains multiple conserved motifs connected with the ability to catalyze the hydrolysis of difficult tertiary ester substrates. Herein, we expanded a library of fluorogenic ester substrates to include more tertiary and constrained esters and screened 33 fluorogenic substrates for activation by LipN, identifying its unique substrate signature. LipN preferred short, unbranched ester substrates, but had its second highest activity against a heteroaromatic five-membered oxazole ester. Oxazole esters are present in multiple mycobacterial serine hydrolase inhibitors but have not been tested widely as ester substrates. Combined structural modeling, kinetic measurements, and substitutional analysis of LipN showcased a fairly rigid binding pocket preorganized for catalysis of short ester substrates. Substitution of diverse amino acids across the binding pocket significantly impacted the folded stability and catalytic activity of LipN with two conserved motifs (HGGGW and GDSAG) playing interconnected, multidimensional roles in regulating its substrate specificity. Together this detailed substrate specificity profile of LipN illustrates the complex interplay between structure and function in mycobacterial hormone-sensitive lipase homologues and indicates oxazole esters as promising inhibitor and substrate scaffolds for mycobacterial hydrolases.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), uses a battery of lipases to scavenge host cell lipids and maintain an infection in its host. With their central roles in infection, lipases have become a viable target for drug development, but many of the lipases in M. tuberculosis remain poorly characterized. Our goal was to determine the substrate specificity, biochemical properties, and potential structural information of LipN, a proposed mycobacterial lipase. Initially, wild type LipN was heterologously expressed, purified to homogeneity, and its substrate specificity characterized against a library of 35 fluorogenic ester substrates. Wild type LipN demonstrated the highest catalytic efficiency against small carbon esters, including methyl ether, ethyl ether, and oxazole esters. LipN's preference for short (three carbons or less) esters with polar substituents suggests its physiological role as an esterase rather than a lipase and a selectivity for polar metabolites. Substitutional mutagenesis across the modeled active site and binding pocket of LipN allowed identification of the essential catalytic serine and histidine residues with the catalytic aspartate playing a less essential role in catalysis. The rest of the binding pocket showcased a range of polar residues required for full catalytic activity and two conserved glycines as the oxyanion hole. Herein, we showed that LipN likely catalyzes the ester hydrolysis of small, polar metabolites potentially aiding in the acquisition of additional energy sources from a host.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), uses a battery of lipases to scavenge host cell lipids and maintain an infection in its host. With their central roles in infection, lipases have become a viable target for drug development, but many of the lipases in M. tuberculosis remain poorly characterized. Our goal was to determine the substrate specificity, biochemical properties, and potential structural information of LipN, a proposed mycobacterial lipase. Initially, wild type LipN was heterologously expressed, purified to homogeneity, and its substrate specificity characterized against a library of 35 fluorogenic ester substrates. Wild type LipN demonstrated the highest catalytic efficiency against small carbon esters, including methyl ether, ethyl ether, and oxazole esters. LipN’s preference for short (three carbons or less) esters with polar substituents suggests its physiological role as an esterase rather than a lipase and a selectivity for polar metabolites. Substitutional mutagenesis across the modeled active site and binding pocket of LipN allowed identification of the essential catalytic serine and histidine residues with the catalytic aspartate playing a less essential role in catalysis. The rest of the binding pocket showcased a range of polar residues required for full catalytic activity and two conserved glycines as the oxyanion hole. Herein, we showed that LipN likely catalyzes the ester hydrolysis of small, polar metabolites potentially aiding in the acquisition of additional energy sources from a host.