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.
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.
Abstract only
Tuberculosis (TB) remains one of the most prevalent diseases in the world, infecting one third of the world's population. The causative agent,
Mycobacterium tuberculosis,
relies on a ...plethora of lipases to maintain an infection in its host. With their central roles in infection, lipases have become a viable target for drug development, especially as drug targets for the dormant state of
M. tuberculosis
. This research aimed to determine the substrate specificity, biochemical properties, and potential structural information of LipN, one proposed mycobacterial lipase. Initially, wild type LipN was expressed in
E. coli
, purified to homogeneity, and its substrate specificity characterized against a library of 35 latent fluorophore substrates. Wild type LipN demonstrated the highest catalytic efficiency against small carbon chains with a k
cat
/K
m
over 10
5
M
−1
s
−1
for its best single acetyl ester substrate and maintained high activity (k
cat
/K
m
> 10
4
) with small polar groups, including esters and oxazole rings. LipN's preference for short nonpolar substrates (three carbons or less) and polar substituents suggests its physiological role as an esterase rather than a lipase. This specificity also suggests hydrogen‐bonding capabilities within the active site of LipN. To categorize the structural factors controlling this substrate specificity, nine variants of LipN were made with substitutions in predicted binding pocket and active site residues and their relative kinetics against the top three ester substrates were characterized. Based on this analysis, the serine and histidine residues of the catalytic triad were positively identified, with 100‐fold decreases in catalytic activity upon their substitution. The aspartate residue in the catalytic triad was tentatively assigned, as multiple aspartate substituted variants showed similar reductions in catalytic efficiency. Future work will be aimed at definitely assigning the catalytic triad as well obtaining a three‐dimensional structure of LipN to confirm the structural features controlling its strict substrate specificity.
Support or Funding Information
Funded by NIH 1R15GM110641‐01A1
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.
Abstract only
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.
PDF
