Comprehensive Substrate Specificity Map Of The Mycobacterial Serine Nhydrolase, Lipn
FASEB JOURNAL(2021)
Abstract
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.
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Key words
mycobacterial serine hydrolase,comprehensive substrate specificity map,substrate specificity,lipn
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