title: A family 11 xylanase from Penicillium funiculosum is strongly inhibited by three wheat xylanase inhibitors.
abstract: Steady-state kinetic approaches were used to investigate the binding of a novel Penicillium funiculosum xylanase, XYNC, with three known xylanase inhibitor proteins from wheat (Triticum aestivum). The xylanase gene (xynC) was cloned from a P. funiculosum genomic library and the deduced amino acid sequence of XYNC exhibited high sequence similarity with fungal family 11 xylanases. xynC was overexpressed in P. funiculosum and the product (XYNC: M(r)=23.6 kDa; pI=3.7) purified and shown to efficiently degrade birchwood xylan [K(m)=0.47% w/v, Vmax=2540 micromol xylose min(-1) (mg protein)(-1) at pH 5.5 and 30 degrees C] and soluble wheat arabinoxylans [K(m)=1.45% w/v, Vmax=7190 micromol xylose min(-1) mg protein)(-1) at pH 5.5 and 30 degrees C]. The xylanase activity of XYNC was inhibited strongly by three xylanase inhibitor proteins from wheat; XIP-I, TAXI I and TAXI II. The inhibition for each was competitive, with very tight binding (K(i)=3.4, 16 and 17 nM, respectively) equivalent to free energy changes (deltaG degrees ) of -49, -45 and -45 kJ mol(-1). This is the first report describing a xylanase that is inhibited by all three wheat xylanase inhibitor proteins described to date.
authors.affiliation: Nutrition and Consumer Sciences Division, Institute of Food Research, Norwich Research Park, Colney, Norwich, UK.
authors.forename: Caroline S M
authors.initials: CS
authors.lastname: Furniss
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Belshaw
authors.forename: Marcos J C
authors.initials: MJ
authors.lastname: Alcocer
authors.forename: Gary
authors.initials: G
authors.lastname: Williamson
authors.forename: Giles O
authors.initials: GO
authors.lastname: Elliott
authors.forename: Kurt
authors.initials: K
authors.lastname: Gebruers
authors.forename: Nigel P
authors.initials: NP
authors.lastname: Haigh
authors.forename: Neville M
authors.initials: NM
authors.lastname: Fish
authors.forename: Paul A
authors.initials: PA
authors.lastname: Kroon
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D005822:Genetic Vectors; D007700:Kinetics; D010407:Penicillium / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014908:Triticum / Q000737:chemistry*; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D017931:DNA Primers; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase