Pectate lyase from Fusarium solani f. sp. pisi: purification, characterization, in vitro translation of the mRNA, and involvement in pathogenicity.
Since indirect experimental evidence suggested that penetration of Fusarium solani f. sp. pisi into its host (Pisum sativum) involved pectin-degrading enzymes (W. Köller, C. R. Allan, and P. E. Kolattukudy (1982) Physiol, Plant Pathol. 20, 47-60), direct tests were made for the production of such degradative enzymes by this pathogen. When the organism was grown on pectin, a pectate lyase (EC 4.2.2.2) was released into the media. This lyase was purified to apparent homogeneity from the culture filtrate by a two-step process involving passage through DEAE-Sephacel followed by hydrophobic interaction chromatography on octyl-Sepharose. The enzyme cleaved polygalacturonate chains in an endo fashion. The molecular mass of the mature extracellular form of this enzyme was estimated to be 26 kDa. The isoelectric point of the enzyme was 8.3 and the optimum pH for activity was 9.4. Calcium was required for activity and evidence is presented that calcium probably interacts with the substrate rather than the enzyme. When antibodies prepared against this enzyme were used for Western blot analysis of the extracellular culture fluid, a single band was observed at 26 kDa. Following in vitro translation of poly(A)+ RNA, a 29-kDa precursor polypeptide was precipitated by the antibodies. Antibodies inhibited both the catalytic activity of the enzyme and the ability of the fungus to infect pea stems, strongly suggesting that this lyase is involved in pathogenesis.
Crawford
M S
MS
Institute of Biological Chemistry, Washington State University, Pullman 99164.
Kolattukudy
P E
PE
D000596:Amino Acids / Q000032:analysis; D002118:Calcium / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005670:Fusarium / Q000201:enzymology* / Q000472:pathogenicity; D006603:Hexuronic Acids / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007158:Immunologic Techniques; D007526:Isoelectric Point; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011133:Polysaccharide-Lyases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*
D000596:Amino Acids; D006603:Hexuronic Acids; D046911:Macromolecular Substances; D012333:RNA, Messenger; C007819:galacturonic acid; D011133:Polysaccharide-Lyases; C023911:pectate lyase; D002118:Calcium