Comparative analysis of the human serine hydrolase OVCA2 to the model serine hydrolase homolog FSH1 from S. cerevisiae

Abstract

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.

Sequence and Structural Motifs Controlling the Broad Substrate Specificity of the Mycobacterial Hormone-Sensitive Lipase LipN

“…37,38 The fluorogenic library is separated into structural groups based on common subclasses of serine hydrolase activity with expansion of the tertiary and bulky esters in the current substrate library (Figure 1). 37,40,45,46 Within these subclasses, the alkyl ester substrates (1−6) allow general classification of serine hydrolases based on substrate length preferences and the introduction of polarity and unsaturation into these substrates (7−11; 12−15) provide greater solubility, reactivity, and selectivity. 38 As a proposed arylesterase with specificity for xenobiotics, 15 LipN may selectively recognize cyclic, hydrophobic, or constrained esters (16)(17)(18)(19)(20)(21)32).…”

“…This pattern of essentiality for the nucleophilic serine and catalytic base histidine and lesser role for the acidic asparate/glutamate residue has been observed in a variety of serine hydrolases and other bHSLs. 40,45,46 Within other conserved bHSL motifs, oxyanion hole residues�Gly 144 and Gly145�were found to be required for complete catalysis, as conservative substitutions with alanine decreased the k cat /K M values by 15-to 140-fold against all three fluorogenic substrates. The only substitution without a near complete loss of catalytic activity was D215A, which had k cat /K M values within 3-fold of wild-type LipN for substrates 1 and 7.…”

“…The enzymatic activity of Mm LipN was measured against p -nitrophenyl acetate, p -nitrophenyl butyrate, p -nitrophenyl valerate, p -nitrophenyl octanoate, and p -nitrophenyl myristate (Sigma-Aldrich) using a 96-well microplate assay (Figure ). ,, Substrates ( p -nitrophenyl acetate (2 M), p -nitrophenyl butyrate (2 M), p -nitrophenyl valerate (2 M), p -nitrophenyl octanoate (200 mM), and p -nitrophenyl myristate (200 mM)) were prepared as stock solutions in acetonitrile and diluted into PBS containing acetylated BSA (PBS–BSA; 0.1 mg/mL). The starting concentration for p -nitrophenyl acetate and p -nitrophenyl butyrate was 20 mM and for p -nitrophenyl valerate, p -nitrophenyl octanoate, and p -nitrophenyl myristate was 2 mM.…”

Effects of mrpigG on Development and Secondary Metabolism of Monascus ruber M7

“…J. Fungi 2020, 6, x FOR PEER REVIEW 13 of 15 hydrolase fold and employ a Ser-His-Asp catalytic triad [21], but more than half of the serine hydrolases (>120 enzymes) remain poorly annotated, with no described physiological function or identified substrates [19,22]. In this study, the results showed that there was a 10-70% decrease in the four typical yellow pigments by mrpigG deletion.…”

“…In current study, the generation of mrpigG mutants by homologous recombination, as shown in Figure 5 and Figure 7 , demonstrated that mrpigG was a member of the MP gene cluster. Searched with the Pfam 33.1 program, the data showed that mrpigG belonged to the serine hydrolase family, which is a large, ubiquitous family of enzymes grouped based on their ability to perform hydrolysis reactions on a range of biological substrates, such as ester, thioester, amide and epoxide bonds in small molecules, peptides or proteins [ 19 , 20 , 21 ]. The majority of serine hydrolases (>60%) adopt an α,β-hydrolase fold and employ a Ser-His-Asp catalytic triad [ 21 ], but more than half of the serine hydrolases (>120 enzymes) remain poorly annotated, with no described physiological function or identified substrates [ 19 , 22 ].…”