A modular esterase from Pseudomonas fluorescens subsp. cellulosa contains a non-catalytic cellulose-binding domain.
The 5' regions of genes xynB and xynC, coding for a xylanase and arabinofuranosidase respectively, are identical and are reiterated four times within the Pseudomonas fluorescens subsp. cellulosa genome. To isolate further copies of the reiterated xynB/C 5' region, a genomic library of Ps. fluorescens subsp. cellulosa DNA was screened with a probe constructed from the conserved region of xynB. DNA from one phage which hybridized to the probe, but not to sequences upstream or downstream of the reiterated xynB/C locus, was subcloned into pMTL22p to construct pFG1. The recombinant plasmid expressed a protein in Escherichia coli, designated esterase XYLD, of M(r) 58,500 which bound to cellulose but not to xylan. XYLD hydrolysed aryl esters, released acetate groups from acetylxylan and liberated 4-hydroxy-3-methoxycinnamic acid from destarched wheat bran. The nucleotide sequence of the XYLD-encoding gene, xynD, revealed an open reading frame of 1752 bp which directed the synthesis of a protein of M(r) 60,589. The 5' 817 bp of xynD and the amino acid sequence between residues 37 and 311 of XYLD were almost identical with the corresponding regions of xynB and xynC and their encoded proteins XYLB and XYLC. Truncated derivatives of XYLD lacking the N-terminal conserved sequence retained the capacity to hydrolyse ester linkages, but did not bind cellulose. Expression of truncated derivatives of xynD, comprising the 5' 817 bp sequence, encoded a non-catalytic polypeptide that bound cellulose. These data indicate that XYLD has a modular structure comprising of a N-terminal cellulose-binding domain and a C-terminal catalytic domain.
Ferreira
L M
LM
Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, U.K.
Wood
T M
TM
Williamson
G
G
Faulds
C
C
Hazlewood
G P
GP
Black
G W
GW
Gilbert
H J
HJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002482:Cellulose / Q000378:metabolism*; D015342:DNA Probes; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017385:Sequence Homology
D015342:DNA Probes; D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D002482:Cellulose; D004950:Esterases