Characterization and sequencing of an active-site cysteine-containing peptide from the xylanase of a thermotolerant Streptomyces.
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.
Keskar
S S
SS
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Rao
M B
MB
Deshpande
V V
VV
D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine / Q000032:analysis*; D004789:Enzyme Activation / Q000187:drug effects; D006026:Glycoside Hydrolases / Q000737:chemistry*; D007202:Indicators and Reagents; D008301:Maleimides; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D010449:Peptide Mapping; D013302:Streptomyces / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000737:chemistry; D043325:Xylan Endo-1,3-beta-Xylosidase; D014990:Xylans / Q000494:pharmacology
D007202:Indicators and Reagents; D008301:Maleimides; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D014990:Xylans; C073040:N-(2,4-dinitroanilino)maleimide; D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase; D003545:Cysteine