Insights into the role of the (alpha+beta) insertion in the TIM-barrel catalytic domain, regarding the stability and the enzymatic activity of chitinase A from Serratia marcescens.
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 on 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 ChiADelta(alpha+beta) was substantially decreased, the pH optimum was shifted from 6.5 to 5.0 and the substrate and product specificities were altered.
Zees
Athanassios C
AC
National and Kapodistrian University of Athens, Department of Biochemistry and Molecular Biology, Panepistimiopolis-Zographou, 15784 Athens, Greece.
Pyrpassopoulos
Serapion
S
Vorgias
Constantinos E
CE
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002688:Chitinases / Q000737:chemistry* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D012706:Serratia marcescens / Q000201:enzymology*; D013057:Spectrum Analysis; D013696:Temperature
D002688:Chitinases