Purification and characterization of an extracellular levansucrase from Pseudomonas syringae pv. phaseolicola.
Levansucrase (EC 2.4.1.10), an exoenzyme of Pseudomonas syringae pv. phaseolicola, was purified to homogeneity from the cell supernatant by chromatography on TMAE-Fraktogel and butyl-Fraktogel. The enzyme has molecular masses of 45 kDa under denaturing conditions and 68 kDa during gel filtration of the native form. In isoelectric focusing, active bands appeared at pH 3.55 and 3.6. Maximum sucrose cleaving activities were measured at pH 5.8 to 6.6 and 60 degrees C. The enzyme was highly tolerant to denaturing agents, proteases, and repeated freezing and thawing. The molecular weight of the produced levan depended on temperature, salinity, and sucrose concentration. The enzyme had levan-degrading activity and did not accept raffinose as a substrate. Comparison of the N-terminal amino acid sequence with the predicted amino acid sequence of levansucrases from Erwinia amylovora and Zymomonas mobilis showed 88 and 69% similarity, respectively, in amino acids 5 to 20. No similarity could be detected to levansucrases of gram-positive bacteria in the first 20 amino acids. By comparison of all levansucrases which have been sequenced to date, the enzyme seems to be conserved in the gram-negative bacteria. The rheological behavior of the product levan prompted a new assessment of the enzyme's role in pathogenesis. Depending on formation conditions, levan solutions exclude other polymer solutions. This behavior supports the presumption that the levansucrase is important in the early phase of infection by creating a separating layer between bacteria and plant cell wall to prevent the pathogen from recognition.
Hettwer
U
U
Institut für Pflanzenpathologie und Pflanzenschutz, Universität Göttingen, Germany.
Gross
M
M
Rudolph
K
K
D000595:Amino Acid Sequence; D005630:Fructans / Q000378:metabolism; D006602:Hexosyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000201:enzymology* / Q000472:pathogenicity; D012212:Rheology; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism; D014774:Virulence
D005630:Fructans; D008670:Metals; D013395:Sucrose; D006602:Hexosyltransferases; C021513:levansucrase