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Evidence for a general role for high‐affinity non‐catalytic cellulose binding domains in microbial plant ceil wall hydroiases
Author(s) -
MillwardSadler S. J.,
Poole D. M.,
Henrissat B.,
Hazlewood G. P.,
Clarke J. H.,
Gilbert H. J.
Publication year - 1994
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1994.tb00317.x
Subject(s) - biology , cellulose , biochemistry , microbiology and biotechnology , computational biology
Summary Cellulases expressed by Cellulomonas fimi consist of a catalytic domain and a discrete non‐catalytic cellulose‐binding domain (CBD). To establish whether CBDs are common features of plant cell‐wall hydroiases from C. fimi , the molecular architecture of xylanase D (XYLD) from this bacterium was investigated. The gene encoding XYLD, designated xynD , consisted of an open reading frame of 1936 bp encoding a protein of M r 68000. The deduced primary sequence of XYLD was confirmed by the size (64kDa) and N‐terminal sequence of the purified recombinant xylanase. Biochemical analysis of the purified enzyme revealed that XYLD is an endo‐acting xylanase which displays no detectable activity against polysaccharides other than xylan. The predicted primary structure of XYLD comprised an /V‐terminal signal peptide followed by a 190‐residue domain that exhibited significant homology to Family‐G xylanases. Truncated derivatives of xynD , encoding the W‐terminal 193 amino acids of mature XYLD directed the synthesis of a functional xylanase, confirming that the 190‐residue N ‐terminal sequence constitutes the catalytic domain. The remainder of the enzyme consisted of two approximately 90‐residue domains, which exhibited extensive homology with each other, and limited sequence identity with CBDs from other polysaccharide hydrolases. Between the two putative CBDs is a 197‐amino‐acid sequence that exhibits substantial homology with Rhizobium NodB proteins. The four discrete domains in XYLD were separated by either threonine/prolineor novel glycine‐rich linker regions. Although full‐length XYLD adsorbed to cellulose, truncated derivatives of the enzyme lacking the C‐terminal CBD hydrolysed xylan but did not bind to cellulose. Fusion of the C‐terminal domain to glutathione‐Stransferase generated hybrid proteins that bound to crystalline cellulose, but not to amorphous cellulose or xylan. The location of CBDs in a C. fimi xylanase indicates that domains of this type are not restricted to cellulases, but are widely distributed between hemicellutases also, and therefore play a pivotal role in the activity of the whole repertoire of plant cell‐wall hydrolases. The role of the NodB homologue in XYLD is less certain.