ABSTRACT
In free-living and parasitic nematodes, the methylation of phosphoethanolamine to phosphocholine provides a key metabolite to sustain phospholipid biosynthesis for growth and development. Because the phosphoethanolamine methyltransferases (PMT) of nematodes are essential for normal growth and development, these enzymes are potential targets of inhibitor design. The pine wilt nematode (Bursaphelenchus xylophilus) causes extensive damage to trees used for lumber and paper in Asia. As a first step toward testing BxPMT1 as a potential nematicide target, we determined the 2.05 Å resolution x-ray crystal structure of the enzyme as a dead-end complex with phosphoethanolamine and S-adenosylhomocysteine. The three-dimensional structure of BxPMT1 served as a template for site-directed mutagenesis to probe the contribution of active site residues to catalysis and phosphoethanolamine binding using steady-state kinetic analysis. Biochemical analysis of the mutants identifies key residues on the ß1d-α6 loop (W123F, M126I, and Y127F) and ß1e-α7 loop (S155A, S160A, H170A, T178V, and Y180F) that form the phosphobase binding site and suggest that Tyr127 facilitates the methylation reaction in BxPMT1.
Subject(s)
Ethanolamines/chemistry , Helminth Proteins/chemistry , Methyltransferases/chemistry , Nematoda/enzymology , Pinus/parasitology , Plant Diseases/parasitology , Amino Acid Sequence , Animals , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Ethanolamines/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Helminth Proteins/genetics , Helminth Proteins/metabolism , Kinetics , Methyltransferases/genetics , Methyltransferases/metabolism , Models, Molecular , Nematoda/genetics , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , ThermodynamicsABSTRACT
Endovascular intervention is a mainstay treatment of peripheral artery disease (PAD) in addition to aggressive risk factor modification and exercise programs in patients with favorable anatomy or in those who are considered too high risk for surgical intervention. Treatment with percutaneous transluminal angioplasty (PTA) and bare metal stents (BMS) has been limited by high rates of in-stent restenosis (ISR) requiring repeat revascularization. Drug-eluting stents (DES), developed and designed to reduce ISR, offer a promising solution to the current challenges in endovascular management of PAD. Several randomized clinical trials have shown improved short- and mid-term outcomes with DES as compared with both PTA and BMS. Herein we provide an up-to-date review of the current literature on DES use in PAD.
