Structural analysis of Bacillus pumilus xylanase

X-ray crystalline analysis of xylanase of Bacillus pumilus was done at 2.2A^^°resolution.Xylanase molecule has a size of 30x40x35A^^°and a cleft observed in the molecule was suggested to be the site of catalytic reaction.Since xylanase and lysozyme catalyze hydrolysis of-1，4-linkage of glycosidic bond，a catalytic mechanism might be similar in both enzymes.Chemical and heavy metal modification of xylanase suggested two acidic amino acid residues as catalytic site.On the other hand，functionally important residues in xylanase will be conserved in those from different origins.Among sequence alignments of 5xylanases derived from Bacillus and fungi，Asp^&lt；21&gt；，Glu^&lt；93&gt；，Asp^&lt；121&gt；and Glu^&lt；182&gt；in B.pumilus xylanase were conserved acidic residues。Asp^&lt；21&gt；was not located in the cleft of the molecule。By taking account the distance between two amino acids，most possible pair as catalytic site was Glu^&lt；93&gt；and Glu^&lt；182&gt；.Before site-directed mutation of the two residues，the region coding signal sequence of the structural gene of xylanase was replaced by the initiation codon using synthetic oligonucleotides.Restriction sites in the structural gene were also modified to create unique sites and ligated downstream of tac promoter in high expression vector in E.coli，pKP 1500.Then，Glu^&lt；93&gt；and Glu^&lt；182&gt；were changed to Asp or Ser.The mutated enzymes were purified to homogeniety from E.coli cell extract.Activity of the mutated enzymes was not detected except the mutation of Glu^&lt；93&gt；to ASP.A little activity observed in Asp^&lt；93&gt；enzyme was the result of marked decrease of Vm value.

X-ray crystalline analysis of xylanase of Bacillus pumilus was done at 2.2 A^^゜ resolution. Xylanase molecule has a size of 30 x 40 x 35 A^^゜ and a cleft observed in the molecule was suggested to be the site of catalytic reaction. Since xylanase and lysozyme catalyze hydrolysis of -1,4-linkage of glycosidic bond, a catalytic mechanism might be similar in both enzymes. Chemical and heavy metal modification of xylanase suggested two acidic amino acid residues as catalytic site. On the other hand, functionally important residues in xylanase will be conserved in those from different origins. Among sequence alignments of 5 xylanases derived from Bacillus and fungi, Asp^<21>, Glu^<93>, Asp^<121> and Glu^<182> in B. pumilus xylanase were conserved acidic residues. Asp^<21> was not located in the cleft of the molecule. By taking account the distance between two amino acids, most possible pair as catalytic site was Glu^<93> and Glu^<182>.Before site-directed mutation of the two residues, the region coding signal sequence of the structural gene of xylanase was replaced by the initiation codon using synthetic oligonucleotides. Restriction sites in the structural gene were also modified to create unique sites and ligated downstream of tac promoter in high expression vector in E. coli, pKP1500. Then, Glu^<93> and Glu^<182> were changed to Asp or Ser. The mutated enzymes were purified to homogeniety from E. coli cell extract. Activity of the mutated enzymes was not detected except the mutation of Glu^<93> to ASP. A little activity observed in Asp^<93> enzyme was the result of marked decrease of Vm value.