1. Structural and biochemical analysis of phosphoethanolamine methyltransferase from the pine wilt nematode Bursaphelenchus xylophilus.
- Author
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Lee SG, Chung MS, DeMarsilis AJ, Holland CK, Jaswaney RV, Jiang C, Kroboth JHP, Kulshrestha K, Marcelo RZW, Meyyappa VM, Nelson GB, Patel JK, Petronio AJ, Powers SK, Qin PR, Ramachandran M, Rayapati D, Rincon JA, Rocha A, Ferreira JGRN, Steinbrecher MK, Yao K, Zhang EJ, Zou AJ, Gang M, Sparks M, Cascella B, Cruz W, and Jez JM
- Subjects
- 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, Thermodynamics, Ethanolamines chemistry, Helminth Proteins chemistry, Methyltransferases chemistry, Nematoda enzymology, Pinus parasitology, Plant Diseases parasitology
- 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 β1d-α6 loop (W123F, M126I, and Y127F) and β1e-α7 loop (S155A, S160A, H170A, T178V, and Y180F) that form the phosphobase binding site and suggest that Tyr127 facilitates the methylation reaction in BxPMT1., Competing Interests: Declaration of Competing Interest The authors have no conflicting interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)
- Published
- 2020
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