Abstract
Four exposed aromatic residues, two in the N-terminal domain (Trp-69 and Trp-33) and two in the catalytic domain (Trp-245 and Phe-232) of Serratia marcescens chitinase A, are linearly aligned with the deep catalytic cleft. To investigate the importance of these residues in the binding activity and hydrolyzing activity against insoluble chitin, site-directed mutagenesis to alanine was carried out. The substitution of Trp-69, Trp-33, or Trp-245 significantly reduced the binding activity to both highly crystalline beta-chitin and colloidal chitin. The substitution of Phe-232, which is located closest to the catalytic cleft, did not affect the binding activity. On the other hand, the hydrolyzing activity against beta-chitin microfibrils was significantly reduced by the substitution of any one of the four aromatic residues including Phe-232. None of the mutations reduced the hydrolyzing activity against soluble substrates. These results clearly demonstrate that the four exposed aromatic residues are essential determinants for crystalline chitin hydrolysis. Three of them, two in the N-terminal domain and one in the catalytic domain, play vital roles in the chitin binding. Phe-232 appeared to be important for guiding the chitin chain into the catalytic cleft. Based on these observations, a model for processive hydrolysis of crystalline chitin by chitinase A is proposed.
Highlights
The chitinases sequenced so far are classified into two different families, families 18 and 19, in the classification system of glycosyl hydrolases based on the amino acid sequence similarity of their catalytic domains [1,2,3]
Four Aromatic Residues Linearly Aligned on the Surface of chitinase A (ChiA) Molecule—ChiA from S. marcescens QMB1466 comprises three domains: an N-terminal domain with an immunoglobulin-like fold, a catalytic (/␣)8 barrel domain, and a small (␣ ϩ ) fold domain, which is inserted in the catalytic (/␣)8 barrel domain [5]
The chitin binding activity of B. circulans chitinase A1 (ChiA1) depends on the C-terminal chitin-binding domain (ChBD) [26]
Summary
3D, three-dimensional; ChiA, S. marcpart by the payment of page charges. This article must be escens chitinase A; ChiA1, B circulans WL-12 chitinase A1; ChBD, hereby marked “advertisement” in accordance with 18 U.S.C. Section chitin-binding domain; CatD, catalytic domain; CatDChiA1, catalytic. Domain of B circulans ChiA1; Cel6A, cellobiohydrolase II; Cel7A, celloʈ To whom correspondence should be addressed. The roles in chitin binding activity and hydrolyzing activity of the two exposed aromatic residues in the catalytic domain and the two additional aromatic residues found on the surface of the N-terminal domain of Serratia ChiA were studied
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