Zees Athanassios C, Pyrpassopoulos Serapion, Vorgias Constantinos E
National and Kapodistrian University of Athens, Department of Biochemistry and Molecular Biology, Panepistimiopolis-Zographou, 15784 Athens, Greece.
Biochim Biophys Acta. 2009 Jan;1794(1):23-31. doi: 10.1016/j.bbapap.2008.09.018. Epub 2008 Oct 10.
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.
粘质沙雷氏菌的几丁质酶A（ChiA）是一种具有高催化活性和高稳定性的嗜温酶。ChiA的晶体结构显示，催化结构域具有TIM桶状折叠结构，在TIM桶状结构的B7β链和A7α螺旋之间有一个（α+β）插入结构，在分子的N端有一个FnIII结构域，以及一个将后者与催化结构域连接起来的铰链区。在本研究中，通过删除该结构域并研究所得截短酶的酶学和结构特性，研究了（α+β）结构域对该酶稳定性、催化活性和特异性的作用。获得的数据清楚地表明，通过去除（α+β）结构域，该酶的热稳定性大幅降低，其表观熔点为42.0±1.0℃，而在pH 9.0时ChiA的表观熔点为58.1±1.0℃。ChiADelta(α+β)的比活性大幅下降，最适pH从6.5变为5.0，底物和产物特异性也发生了改变。
