Conserved serine‐rich sequences in xylanase and cellulase from Pseudomonas fluorescens subspecies cellulosa: internal signal sequence and unusual protein processing

Abstract

The complete nucleotide sequence of the xynA gene coding for a xylanase (XYLA) expressed by Pseudomonas fluorescens subspecies cellulosa, has been determined. The structural gene consists of an open reading frame of 1833 bp followed by a TAA stop codon. Confirmation of the nucleotide sequence was obtained by comparing the predicted amino acid sequence with that derived by N-terminal analysis of purified forms of the xylanase. The signal peptide present at the N terminus of mature XYLA closely resembles signal peptides of other secreted proteins. Truncated forms of the xylanase gene, in which the sequence encoding the N-terminal signal peptide had been deleted, still expressed coli. XYLA contains domains which are homologous to an endoglucanase expressed by the same organism. These structures include serine-rich sequences. Bal31 deletions of xynA revealed the extent to which these conserved sequences, in XYLA, were essential for xylanase activity. Downstream of the TAA stop codon is a G + C-rich region of dyad symmetry (delta G = 24 kcal) characteristic of E. coli Rho-independent transcription terminators.

Structural and functional relationships in two families of β‐1,4‐glycanases

“…Sequence alignment,for /3-1,4-glucanases offamily F. The sequences shown are from the catalytic domains or pulative catalytic domains of (a) Cex, C.,fimi [38]; (b) XynZ, Clostridium thermocellum [26]; (c) XynA, ~seudomonus,fluorescens subsp. cellulosa [39]; (d) CelB, exoglucanase domain, Cuidocellum sacchurolyticum [40]; (e) XynA, C. saccharolyticum [41]; (0 XynA, Bucillus sp. strain C125 [42]; (g) XynA, Butyrivihrio jibrisolvens 1431; (h) ORF4, C'.…”

Calcium Protects a Mesophilic Xylanase from Proteinase Inactivation and Thermal Unfolding

“…The EDTA was then removed by dialysis against 100 volumes of 10 mM Tris/HCl buffer, pH 8.0, at 4°C. XYLA was then purified by anion-exchange chromatography (17). The purified enzymes were dialyzed against 3 ϫ 1,000 volumes of 50 mM Tris/HCl buffer, pH 7.5, containing 10 g/liter Chelex 100 (Bio-Rad) to remove any remaining Ca 2ϩ .…”

“…Aliquots were removed at each time point between 0 and 20 min, heated to 100°C in the presence of 2% SDS, and then subjected to SDS-PAGE (21). The NH 2 -terminal sequence of the major proteolytic product generated (29 kDa) was determined as described previously (17). To determine the precise size of this peptide, the proteolytic reaction was terminated by the addition of phenylmethylsulfonyl fluoride (1 mM) rather than SDS , and the sample was dialyzed extensively against distilled water and then subjected to electrospray ionization mass spectrometry using a VG Quattro Tandem Quadropole mass spectrometer.…”

“…Purification of Wild Type and Mutant Forms of XYLA-Recombinant strains of Escherichia coli expressing XYLA or its derivatives were cultured as in (17). Periplasmic fractions of the cells were prepared in the presence of 25 mM EDTA to complex both free and XYLA-associated calcium.…”

Unusual sequence organization in CenB, an inverting endoglucanase from Cellulomonas fimi

“…Although its role, if any, in the binding of CenB to cellulose was not established, the sequence was designated CBDCenB. Similar sequences occur at the N or C termini of other bacterial cellulases and a xylanase (1,3,13,14,36). The consensus for these sequences is given in Fig.…”