Bile Acid Recognition by NAPE-PLD

Abstract

The membrane-associated enzyme NAPE-PLD (N-acyl phosphatidylethanolamine specific-phospholipase D) generates the endogenous cannabinoid arachidonylethanolamide and other lipid signaling amides, including oleoylethanolamide and palmitoylethanolamide. These bioactive molecules play important roles in several physiological pathways including stress and pain response, appetite and lifespan. Recently, we reported the crystal structure of human NAPE-PLD and discovered specific binding sites for the bile acid deoxycholic acid. In this study we demonstrate that in the presence of this secondary bile acid, the stiffness of the protein measured by elastic neutron scattering increases, and NAPE-PLD results ~7 times faster to catalyze the hydrolysis of the more unsaturated substrate N-arachidonyl-phosphatidylethanolamine, compared with N-palmitoyl-phosphatidylethanolamine. Chenodeoxycholic acid and glyco- or tauro-dihydroxy conjugates can also bind to NAPE-PLD and drive its activation. The only natural monohydroxy bile acid, lithocholic acid, shows an affinity of ~20 μM and acts instead as a reversible inhibitor (IC50 ≈ 68 μM). Overall, these findings provide important insights into the allosteric regulation of the enzyme mediated by bile acid cofactors, and reveal that NAPE-PLD responds primarily to the number and position of their hydroxyl groups.

Symmetrically substituted dichlorophenes inhibit N-acyl-phosphatidylethanolamine phospholipase D

“…LCA is currently the only known endogenous inhibitor of NAPE-PLD (35). There are a variety of primary and secondary bile acids besides LCA and these differ from each other in respect to the number, position, and stereochemistry of their hydroxyl groups as well as their conjugated moieties.…”

“…There is currently a lack of specific inhibitors or activators to directly assess the contribution of NAPE-PLD to NAE biosynthesis under various conditions. One endogenous bile acid, lithocholic acid (LCA), has been reported to inhibit NAPE-PLD (IC 50 68 μM) (35). Although its bioavailability and relatively low toxicity allow LCA to be administered in vivo, LCA is also a potent ligand for the bile acid receptor, Tgr5, with an EC50 of 0.53 μM (36) so it cannot be used as a selective inhibitor of NAPE-PLD.…”

Microbiome, bile acids, and obesity: How microbially modified metabolites shape anti‐tumor immunity

“…34,35 Another receptor-independent function is allosteric activities, where BAs can inactivate or stabilize enzymes. 36 Since gut microbes harbor enzymes that modify BAs hydroxylation configurations, the gut microbiome composition and functions shape circulating BA pools and their signaling mediated affects throughout the body. For instance, the bacteria Clostridium cluster 14 and Bacteroides contain high levels of 7-alpha-hydroxylase, which convert primary BAs to secondary BAs by removing hydroxylation at the 7 alpha carbon position.…”

Synthesis and characterization of the first inhibitor ofN-acylphosphatidylethanolamine phospholipase D (NAPE-PLD)

“…The entry cavity also contains two recognition pockets, one in each monomer, which selectively recognize secondary bile acids such as deoxycholic acid (DCA). 19 This binding event enhances dimer assembly and enables catalysis. 2 The molecular architecture of NAPE-PLD offers two obvious targets for pharmacological modulation: the catalytic zinc center and the allosteric bile acid-binding site (Figure 1).…”