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Sorbitol dehydrogenase from Bacillus subtilis. Purification, characterization, and gene cloning
The Journal of Biological Chemistry
|December 15, 1992
Summary
Researchers cloned the Bacillus subtilis sorbitol dehydrogenase gene (gutB) for protein engineering studies. This enzyme is a tetramer containing zinc, with characterized substrate specificity and potential zinc-binding sites identified through sequence analysis.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Bacillus subtilis sorbitol dehydrogenase (SORD) is an enzyme with potential for studying enzyme mechanisms.
- Understanding SORD's structure and function is crucial for protein engineering applications.
Purpose of the Study:
- To clone the gutB gene encoding B. subtilis SORD for further study.
- To characterize the purified B. subtilis SORD enzyme.
- To identify potential zinc-binding sites and analyze sequence homology.
Main Methods:
- Purification and characterization of B. subtilis SORD.
- Atomic absorption analysis for zinc content.
- Amino acid sequencing and gene cloning using oligonucleotide probes.
- DNA sequencing and sequence alignment with other SORDs and alcohol dehydrogenases.
Main Results:
- B. subtilis SORD was purified as a homogeneous tetrameric enzyme (38 kDa subunits) containing 1 mol zinc/subunit.
- Substrate and stereospecificity towards hexitols were determined.
- The gutB gene was cloned and sequenced, revealing 36% sequence identity with mammalian SORDs.
- Two potential zinc-binding sites were identified by comparing with alcohol dehydrogenase sequences.
Conclusions:
- The cloned gutB gene provides a system for protein engineering of B. subtilis SORD.
- Characterization revealed key properties including zinc content and substrate specificity.
- Sequence analysis identified conserved regions and potential functional sites, aiding structure-function relationship studies.