Mutational analysis of endoxylanases XylA and XylB from the phytopathogen Fusarium graminearum reveals comprehensive insights into their inhibitor insensitivity.
Endo-beta-1,4-xylanases (EC 3.2.1.8; endoxylanases), key enzymes in the degradation of xylan, are considered to play an important role in phytopathogenesis, as they occupy a prominent position in the arsenal of hydrolytic enzymes secreted by phytopathogens to breach the cell wall and invade the plant tissue. Plant endoxylanase inhibitors are increasingly being pinpointed as part of a counterattack mechanism. To understand the surprising XIP-type endoxylanase inhibitor insensitivity of endoxylanases XylA and XylB from the phytopathogen Fusarium graminearum, an extensive mutational study of these enzymes was performed. Using combinatorial and site-directed mutagenesis, the XIP insensitivity of XylA as well as XylB was proven to be solely due to amino acid sequence adaptations in the "thumb" structural region. While XylB residues Cys141, Asp148, and Cys149 were shown to prevent XIP interaction, the XIP insensitivity of XylA could be ascribed to the occurrence of only one aberrant residue, i.e., Val151. This study, in addition to providing a thorough explanation for the XIP insensitivity of both F. graminearum endoxylanases at the molecular level, generated XylA and XylB mutants with altered inhibition specificities and pH optima. As this is the first experimental elucidation of the molecular determinants dictating the specificity of the interaction between endoxylanases of phytopathogenic origin and a plant inhibitor, this work sheds more light on the ongoing evolutionary arms race between plants and phytopathogenic fungi involving recognition of endoxylanases.
Beliën
Tim
T
Laboratory of Gene Technology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium. tim.belien@biw.kuleuven.be
Van Campenhout
Steven
S
Van Acker
Maarten
M
Robben
Johan
J
Courtin
Christophe M
CM
Delcour
Jan A
JA
Volckaert
Guido
G
D019943:Amino Acid Substitution / Q000235:genetics; D004252:DNA Mutational Analysis; D025141:Drug Resistance, Fungal / Q000235:genetics*; D043364:Endo-1,4-beta Xylanases / Q000037:antagonists & inhibitors* / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004795:Enzyme Stability / Q000235:genetics; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D005670:Fusarium / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed
D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D043364:Endo-1,4-beta Xylanases