Journal
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
Volume 1794, Issue 1, Pages 23-31Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbapap.2008.09.018
Keywords
Chitinase; Serratia; TIM-barrel insertion; Enzyme engineering; Protein modelling; Thermodynamics
Categories
Funding
- European Scientific Network
- Graduate Fellowship Program of CSR Demokritos
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Chitinase A (ChiA) from Serratia marcescens is a mesophilic enzyme with high catalytic activity and high stability. The crystal structure of ChiA has revealed a TIM-barrel fold of the catalytic domain, an (alpha+beta) insertion between the B7 beta-strand and A7 alpha-helix of the TIM-barrel, an FnIII domain at the N-terminus of the molecule and a hinge region that connects the latter to the catalytic domain. In this study, the role of the (alpha+beta) domain oil the stability, catalytic activity and specificity of the enzyme was investigated by deleting this domain and studying the enzymatic and structural properties of the resulting truncated enzyme. The obtained data clearly show that by removing the (alpha+beta) domain, the thermal stability of the enzyme is substantially reduced, with an apparent T-m of 42.0 +/- 1.0 degrees C, compared to the apparent T-m of 58.1 +/- 1.0 degrees C of ChiA at pH 9.0. The specific activity of ChiA Delta(alpha+beta) Was Substantially decreased, the pH optimum was shifted from 6.5 to 5.0 and the Substrate and product specificities were altered. (C) 2008 Elsevier B.V. All rights reserved.
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