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A modular xylanase containing a novel non-catalytic xylan-specific binding domain
G W Black1, G P Hazlewood, S J Millward-Sadler
1Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, U.K.
The Biochemical Journal
|April 1, 1995
Summary
Xylanase D (XYLD) features an internal domain that binds xylan, enhancing affinity for insoluble xylan. This domain influences binding but not catalytic activity, offering evolutionary insights.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Xylanase D (XYLD) from Cellulomonas fimi possesses both catalytic and binding domains.
- The enzyme contains a C-terminal cellulose-binding domain (CBD) and an internal domain with homology to CBDs.
Purpose of the Study:
- To investigate the function of XYLD's domains in substrate binding and catalytic activity.
- To determine the role of the internal CBD homologue in xylan and cellulose binding.
Main Methods:
- Site-directed mutagenesis to create truncated XYLD variants lacking specific CBDs.
- Enzyme kinetic assays (Km, specific activity) using soluble and insoluble xylan.
- Polysaccharide binding assays using purified proteins and fusion proteins.
Main Results:
- The C-terminal CBD is essential for cellulose binding; its deletion retains xylan binding.
- The internal CBD homologue is responsible for xylan binding.
- Both domains are required for binding to both cellulose and xylan.
- The internal xylan-binding domain significantly lowers the Km for insoluble xylan but does not affect catalytic activity.
Conclusions:
- XYLD possesses a distinct internal xylan-binding domain.
- This domain enhances enzyme affinity for insoluble xylan substrates.
- The internal domain's primary role is substrate recognition and affinity, not catalysis.