Structural and functional role of tryptophan in xylanase from an extremophilic Bacillus: assessment of the active site.
Microenvironment and conformation of the active site of xylanase from an extremophilic Bacillus was deciphered for the first time using fluorescence spectroscopy. NBS modified enzyme showed complete inactivation and the kinetic analysis implicated the presence of an essential tryptophan at the active site of xylanase. Xylan (0.5%) protected the enzyme completely from inactivation with NBS, whereas it afforded 35% protection against the loss of fluorescence, suggesting that not all the tryptophans are involved at the substrate binding site. Quenching studies revealed that acrylamide was more efficient than KI and CsCl as indicated by the higher Stern-Volmer quenching constants (Ksv). The steric factor represented by the percentage accessibility of the tryptophan residues of XylII was higher with the positively charged Cs+ (80) than with the negatively charged I- (10), suggesting that the tryptophan residues are located in a relatively electronegative environment. In the presence of 6 M Gdn HCl the fluorescence shifted to 350 nm with increased accessibility of the fluorophore to the quenchers. The proximity of the essential carboxyl groups with a high pKa value of 6.9 [Chauthaiwale and Rao (1994) Biochim. Biophys. Acta] probably contributes to the electronegative environment of the tryptophan residue. Our results on sequence analysis of the gene encoding for XylII (Accession Number U83602 in the GenBank database) have shown that Trp 61 is highly conserved and may play a role in the structure-function relationship of the enzyme.
Nath
D
D
Division of Biochemical Sciences, National Chemical Laboratory, Pashan, Pune, India.
Rao
M
M
D001407:Bacillus / Q000201:enzymology*; D004789:Enzyme Activation; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry* / Q000378:metabolism*
D014364:Tryptophan; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase