Characterization and sequencing of an active-site cysteine-containing peptide from the xylanase of a thermotolerant Streptomyces

Abstract

The kinetics of chemical modification of the xylanase from a thermotolerant Streptomyces T7 indicated the involvement of 1 mol of cysteine residue/mol of enzyme [Keskar, Srinivasan & Deshpande (1989) Biochem. J. 261, 49-55]. The chromophoric reagent N-(2,4-dinitroanilino)maleimide (DAM) reacts covalently with thiol groups of xylanase with complete inactivation. Protection against inactivation was provided by the substrate (xylan). The purified xylanase that had been modified with DAM was digested with pepsin and the peptides were purified by gel filtration followed by peptide mapping. The active-site peptide was distinguished from the other thiol-containing peptides by comparison of the peptides generated by labelling the enzyme in the presence and in the absence of the substrate. The peptide mapping of the modified enzyme in the absence of xylan showed three yellow peptides, whereas in the presence of xylan only two yellow peptides were detected. The active-site peptide protected by the substrate failed to form the complex with DAM. The modified active-site peptide was isolated and sequenced. Gas-phase sequencing provided the following sequence: Ser-Val-Ile-Met-Xaa-Ile-Asp-His-Ile-Arg-Phe. This is the first report on the isolation and sequencing of the active-site peptide from a xylanase. The comparison of reactive cysteine-containing peptide sequence with the catalytic regions of other glucanases revealed the presence of a conserved aspartic acid residue.

Microbial xylanases and their industrial application in pulp and paper biobleaching: a review

“…Wheat bran was found to be the best substrate for xylanase production by alkalophilic Paenibacillus polymyxa CKWX1 (Walia et al 2013b) and alkalophilic Streptomyces T-7 (Keskar et al 1992). The highest levels of xylanase were formed when Cellulosimicrobium cellulans CKMX1 was grown on apple pomace (Walia et al 2013a), corn cob (Purkarthofer et al 1993), sawdust (Yu et al 1997), sugar beet pulp (Tuohy et al 1993) and sugarcane bagasse (Bocchini et al 2005).…”

Molecular and biotechnological aspects of xylanases

“…The characterization and sequencing of the cysteine containing active site peptide of the xylanase from Streptomyces T-7 [171] and Chainia [172] have been reported. The characterization and sequencing of the cysteine containing active site peptide of the xylanase from Streptomyces T-7 [171] and Chainia [172] have been reported.…”

“…Two L-sheets are twisted around a deep, long cleft which is lined with many aromatic residues and is large enough to accommodate at least four xylose residues. The hydrogen bond between Tyr 171 and Tyr 77 exists in other xylanases although the tyrosine residue is substituted by histidine in a few cases. The enzymes of family 11 are single domain proteins composed of three antiparallel L-sheets and one K-helix, with the active site lying between the second and third sheets.…”

Xylanases: from Biology to BioTechnology

“…(J 981 ) indicate that ionizable groups participate in the catalysis in a xylanase frolll A. niger and several reports sho\v that carboxy groups are involved (Bray and Clarke, (990: Zhu (~t al. .. 1994)~second, several studies with the fUllctionaJ)y related enzylne, ccllulase~suggest the invol vetnent of catalytic carboxy groups (Hurst, Sullivan and Shepherd, 1977~Yaguchi el lIl., 1983~Paice et al, 1984Clarke and Yaguchi.. 1985;H9Sj et al, 1989;Chuuvaux, Beguin and Aubert, 1992) and third, several groups succeeded in lllapping both essential carboxy anlino acid residues by utilizing a nlunber of independent experinlental approaches (Okada, 1989: Tu1l el {II.,J991; Chauvaux, Beguin and Aubert, 1992;Keskar, Rao and Deshpande, 1992;Ko elll/.• 1992; Lee el (ll.~t 993: Bray and Clarke, 1994: Macleod et (II., 1994~Miao el al., 1994 T()rronen~Harkki and Rouvinen, ]994; Moreau el ld., J994a~Wakarchuk el al.1 994(1).…”