A single domain thermophilic xylanase can bind insoluble xylan: evidence for surface aromatic clusters.
A clone expressing xylanase activity in Escherichia coli has been selected from a genomic plasmid library of the thermophilic Bacillus strain D3. Subcloning from the 9-kb insert located the xylanase activity to a 2.7-kb HindII/BamHI fragment. The DNA sequence of this clone revealed an ORF of 367 codons encoding a single domain type-F or family 10 enzyme, which was designated as XynA. Purification of the enzyme following over-expression in E. coli produced an enzyme of 42 kDa with a temperature optimum of 75 degrees C which can efficiently bind and hydrolyse insoluble xylan. The pH optimum of the enzyme is 6.5, but it is active over a broad pH range. A homology model of the xylanase has been constructed which reveals a series of surface aromatic residues which form hydrophobic clusters. This unusual structural feature is strikingly similar to the situation observed in the structure determined for the type-G xylanase from the Bacillus D3 strain and may constitute a common evolutionary mechanism imposed on different structural frameworks by which these xylanases may bind potential substrates and exhibit thermostability.
Connerton
I
I
Division of Food Science, School of Biological Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK. ian.connerton@nottingham.ac.uk
Cummings
N
N
Harris
G W
GW
Debeire
P
P
Breton
C
C
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D016415:Sequence Alignment; D013696:Temperature; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase