title: Evidence for a general role for non-catalytic thermostabilizing domains in xylanases from thermophilic bacteria.
abstract: A genomic library of Clostridium thermocellum DNA constructed in lambda ZAPII was screened for xylanase-expressing clones. Cross-hybridization experiments revealed a new xylanase gene isolated from the gene library, which was designated xyn Y. The encoded enzyme, xylanase Y (XYLY), displayed features characteristic of an endo-beta1,4-xylanase: the enzyme rapidly hydrolysed oat spelt, wheat and rye arabinoxylans and was active against methyl-umbelliferyl-beta-D-cellobioside, but did not hydrolyse any cellulosic substrates. The pH and temperature optima of the enzyme were 6.8 and 75 degrees C respectively, and the recombinant XYLY, expressed by Escherichia coli had a maximum Mr of 116000. The nucleotide sequence of xyn Y contained an open reading frame of 3228 bp encoding a protein of predicted Mr 120 105. The encoded enzyme contained a typical N-terminal 26-residue signal peptide, followed by a 164 amino acid sequence, designated domain A, that was not essential for catalytic activity. Downstream of domain A was a 351-residue xylanase Family F catalytic domain, followed by a 180-residue sequence that exhibited 28% sequence identity with a thermostable domain of Thermoanaerobacterium saccharolyticum xylanase A. The C-terminal portion of XYLY comprised the 23-residue duplicated docking sequence found in all other C. thermocellum plant cell wall hydrolases that are constituents of the bacterium's multienzyme complex, termed the cellulosome, followed by a 286-residue domain which exhibited 32% sequence identity with the N-terminal region of C. thermocellum xylanase Z. The enzyme did not contain linker sequences found in other C. thermocellum plant cell wall hydrolases. Analysis of truncated forms of XYLY and hybrid proteins, comprising segments of XYLY fused to the E. coli maltose binding domain, confirmed that XYLY contained a central catalytic domain and an adjacent thermostable domain. The C-terminal domain did not bind to cellulose or xylan. Western blot analysis using antiserum raised against XYLY showed that the xylanase was located in the cellulosome and did not appear to be extensively glycosylated. The non-catalytic domains of XYLY are discussed in relation to the general stability of thermophilic xylanases.
authors.affiliation: Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, U.K.
authors.forename: C M
authors.initials: CM
authors.lastname: Fontes
authors.forename: G P
authors.initials: GP
authors.lastname: Hazlewood
authors.forename: E
authors.initials: E
authors.lastname: Morag
authors.forename: J
authors.initials: J
authors.lastname: Hall
authors.forename: B H
authors.initials: BH
authors.lastname: Hirst
authors.forename: H J
authors.initials: HJ
authors.lastname: Gilbert
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D043364:Endo-1,4-beta Xylanases; D015723:Gene Library; D005798:Genes, Bacterial; D006093:Gram-Positive Asporogenous Rods / Q000201:enzymology*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases