Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.1.5 (neuropathy target esterase)
 1,070 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Secreted phospholipase B enzymes (PLB1) with high levels of N-linked glycosylation are proven fungal virulence determinants. We demonstrated that removal of N-linked glycans from secreted cryptococcal PLB1 leads to loss of enzyme activity. To determine if individual N-glycan attachment sites affect secretion of active enzyme, we altered three along the entire length of the protein, by site-directed mutagenesis, namely Asn56, Asn430 and Asn550 to Ala, in wild-type PLB1 (full length) and a glycosylphosphatidylinositol (GPI) anchorless version (PLB1(GPI-)) that is hypersecreted due to lack of membrane association. Alteration of Asn56 and Asn550 in both PLB1 and PLB1(GPI-) abolished enzyme secretion while alteration of Asn430 reduced secretion by 60%, following expression in Saccharomyces cerevisiae. Reduced secretion coincided with reduced enzyme in membranes and cell walls confirming a reduction in the rate of PLB1 transport to the cell surface. Deglycosylation of cryptococcal PLB1 increased its susceptibility to proteolysis suggesting that the absence of full glycosylation status leads to degradation of unstable PLB1, resulting in reduced traffic through the secretory pathway. We conclude that individual N-linked glycans are required for optimal transport of PLB1 to the cell surface and optimal secretion of both PLB1 and PLB1(GPI-).
 ...
PMID:N-linked glycosylation sites affect secretion of cryptococcal phospholipase B1, irrespective of glycosylphosphatidylinositol anchoring. 1691 92

Phospholipase B1 (PLB1), secreted by the pathogenic yeast Cryptococcus neoformans, has an established role in virulence. Although the mechanism of its phospholipase B, lysophospholipase, and lysophospholipase transacylase activities is unknown, it possesses lipase, subtilisin protease aspartate, and phospholipase motifs containing putative catalytic residues S146, D392, and R108, respectively, conserved in fungal PLBs and essential for human cytosolic phospholipase A2 (cPLA2) catalysis. To determine the role of these residues in PLB1 catalysis, each was substituted with alanine, and the mutant cDNAs were expressed in Saccharomyces cerevisiae. The mutant PLB1s were deficient in all three enzymatic activities. As the active site structure of PLB1 is unknown, a homology model was developed, based on the X-ray structure of the cPLA2 catalytic domain. This shows that the two proteins share a closely related fold, with the three catalytic residues located in identical positions as part of a single active site, with S146 and D392 forming a catalytic dyad. The model suggests that PLB1 lacks the "lid" region which occludes the cPLA2 active site and provides a mechanism of interfacial activation. In silico substrate docking studies with cPLA2 reveal the binding mode of the lipid headgroup, confirming the catalytic dyad mechanism for the cleavage of the sn-2 ester bond within one of two separate binding tracts for the lipid acyl chains. Residues specific for binding arachidonic and palmitic acids, preferred substrates for cPLA2 and PLB1, respectively, are identified. These results provide an explanation for differences in substrate specificity between lipases sharing the cPLA2 catalytic domain fold and for the differential effect of inhibitors on PLB1 enzymatic activities.
 ...
PMID:Role of conserved active site residues in catalysis by phospholipase B1 from Cryptococcus neoformans. 1768 90

Escherichia coli possesses a versatile protein with the enzyme activities of thioesterase I, protease I, and lysophospholipase L(1). The protein is dubbed as TAP according to the chronological order of gene discovery (TesA/ApeA/PldC). Our previous studies showed that TAP comprises the catalytic triad Ser(10), Asp(154), and His(157) as a charge relay system, as well as Gly(44) and Asn(73) residues devoted to oxyanion hole stabilization. Geometrically, about 10 A away from the enzyme catalytic cleft, Trp(23) showed a stronger resonance shift than the backbone amide resonance observed in the nuclear magnetic resonance (NMR) analyses. In the present work, we conducted site-directed mutagenesis to change Trp into alanine (Ala), phenylalanine (Phe), or tyrosine (Tyr) to unveil the role of the Trp(23) indole ring. Biochemical analyses of the mutant enzymes in combination with TAP's three-dimensional structures suggest that by interlinking the residues participating in this catalytic machinery, Trp(23) could effectively influence substrate binding and the following turnover number. Moreover, it may serve as a contributor to both H-bond and aromatic-aromatic interaction in maintaining the cross-link within the interweaving framework of protein.
 ...
PMID:Functional role of a non-active site residue Trp(23) on the enzyme activity of Escherichia coli thioesterase I/protease I/lysophospholipase L(1). 1954 Mar 68

In Bacillus subtilis, the germination-related lipase LipC is located in the spore coat, and mutant spores are defective in L-alanine-stimulated germination. To determine the physiological role of LipC, the recombinant LipC expressed in Escherichia coli was purified and characterized. The enzyme hydrolyzes p-nitrophenyl ester substrates with various acyl-chain lengths. Thin-layer chromatography and gas chromatography-mass spectrometry analysis indicated that LipC cleaves the fatty acids at the sn-1 and sn-2 positions of phospholipids as phospholipase B, and that the enzyme shows no selectivity for the polar head groups of lipid molecules. When the amounts of free fatty acids in dormant wild-type and lipC mutant (YCSKd) spores were measured, the amount of free fatty acids in the YCSKd spores was about 35% less than in the wild-type spores. These results suggest the possibility that Bacillus subtilis LipC plays an important role in the degradation of the outer spore membrane during sporulation.
 ...
PMID:Bacillus subtilis spore coat protein LipC is a phospholipase B. 2005 19


<< Previous 1 2