Abstract:
:The retaining beta-1,4-glycanase Cex from Cellulomonas fimi, a family 10 glycosyl hydrolase, hydrolyzes xylan 40-fold more efficiently than cellulose. To gain insight into the nature of its preference for xylan, we determined the crystal structure of the Cex catalytic domain (Cex-cd) trapped as its covalent 2-deoxy-2-fluoroxylobiosyl-enzyme intermediate to 1.9 A resolution. Together with the crystal structure of unliganded Cex-cd [White, A., et al. (1994) Biochemistry 33, 12546-12552] and the previously determined crystal structure of the covalent 2-deoxy-2-fluorocellobiosyl-Cex-cd intermediate [White, A., et al. (1996) Nat. Struct. Biol. 3, 149-154], this structure provides a convincing rationale for the observed substrate specificity in Cex. Two active site residues, Gln87 and Trp281, are found to sterically hinder the binding of glucosides and must rearrange to accommodate these substrates. Such rearrangements are not necessary for the binding of xylobiosides. The importance of this observation was tested by examining the catalytic behavior of the enzyme with Gln87 mutated to Met. This mutation had no measurable effect on substrate affinity or turnover number relative to the wild type enzyme, indicating that the Met side chain could accommodate the glucoside moiety as effectively as the wild type Gln residue. Subsequent mutagenesis studies will address the role of entropic versus enthalpic contributions to binding by introducing side chains that might be more rigid in the unliganded enzyme.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Notenboom V,Birsan C,Warren RA,Withers SG,Rose DRdoi
10.1021/bi9729211subject
Has Abstractpub_date
1998-04-07 00:00:00pages
4751-8issue
14eissn
0006-2960issn
1520-4995pii
bi9729211journal_volume
37pub_type
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