A family 11 xylanase from Penicillium funiculosum is strongly inhibited by three wheat xylanase inhibitors.
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.
Furniss
Caroline S M
CS
Nutrition and Consumer Sciences Division, Institute of Food Research, Norwich Research Park, Colney, Norwich, UK.
Belshaw
Nigel J
NJ
Alcocer
Marcos J C
MJ
Williamson
Gary
G
Elliott
Giles O
GO
Gebruers
Kurt
K
Haigh
Nigel P
NP
Fish
Neville M
NM
Kroon
Paul A
PA
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
D017931:DNA Primers; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase