A MODULAR ESTERASE FROM PSEUDOMONAS-FLUORESCENS SUBSP CELLULOSA CONTAINS A NONCATALYTIC CELLULOSE-BINDING DOMAIN

The 5' regions of genes xynB and xynC, coding for a xylanase and arabi nofuranosidase respectively, are identical and are reiterated four tim es within the Pseudomonas fluorescens subsp. cellulosa genome. To isol ate further copies of the reiterated xynB/C 5' region, a genomic libra ry of Ps. fluorescens subsp. cellulosa DNA was screened with a probe c onstructed 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 construc t pFG1. The recombinant plasmid expressed a protein in Escherichia col i, designated esterase XYLD, of M(r) 58 500 which bound to cellulose b ut not to xylan. XYLD hydrolysed aryl esters, released acetate groups from acetylxylan and liberated 4-hydroxy-3-methoxycinnamic acid from d estarched wheat bran. The nucleotide sequence of the XYLD-encoding gen e, 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 a mino acid sequence between residues 37 and 311 of XYLD were almost ide ntical with the corresponding regions of xynB and xynC and their encod ed 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 derivati ves of xynD, comprising the 5' 817 bp sequence, encoded a non-catalyti c polypeptide that bound cellulose. These data indicate that XYLD has a modular structure comprising of a N-terminal cellulose-binding domai n and a C-terminal catalytic domain.