Department of Biology, University of Waterloo, Waterloo, Ontario, Canada, N2L 3G1.
Protein Sci. 2014 May;23(5):586-93. doi: 10.1002/pro.2437. Epub 2014 Mar 10.
Maize ChitA chitinase is composed of a small, hevein-like domain attached to a carboxy-terminal chitinase domain. During fungal ear rot, the hevein-like domain is cleaved by secreted fungal proteases to produce truncated forms of ChitA. Here, we report a structural and biochemical characterization of truncated ChitA (ChitA ΔN), which lacks the hevein-like domain. ChitA ΔN and a mutant form (ChitA ΔN-EQ) were expressed and purified; enzyme assays showed that ChitA ΔN activity was comparable to the full-length enzyme. Mutation of Glu62 to Gln (ChitA ΔN-EQ) abolished chitinase activity without disrupting substrate binding, demonstrating that Glu62 is directly involved in catalysis. A crystal structure of ChitA ΔN-EQ provided strong support for key roles for Glu62, Arg177, and Glu165 in hydrolysis, and for Ser103 and Tyr106 in substrate binding. These findings demonstrate that the hevein-like domain is not needed for enzyme activity. Moreover, comparison of the crystal structure of this plant class IV chitinase with structures from larger class I and II enzymes suggest that class IV chitinases have evolved to accommodate shorter substrates.
玉米几丁质酶 A 由一个小的海豆荚球蛋白样结构域连接到羧基末端几丁质酶结构域组成。在真菌穗腐病期间，海豆荚球蛋白样结构域被分泌的真菌蛋白酶切割，产生 ChitA 的截断形式。在这里，我们报告了截断的 ChitA（ChitA ΔN）的结构和生化特性，它缺乏海豆荚球蛋白样结构域。ChitA ΔN 和一种突变形式（ChitA ΔN-EQ）被表达和纯化；酶活性测定表明，ChitA ΔN 的活性与全长酶相当。将 Glu62 突变为 Gln（ChitA ΔN-EQ）消除了几丁质酶活性，而不破坏底物结合，表明 Glu62 直接参与催化。ChitA ΔN-EQ 的晶体结构为 Glu62、Arg177 和 Glu165 在水解中的关键作用以及 Ser103 和 Tyr106 在底物结合中的作用提供了强有力的支持。这些发现表明，海豆荚球蛋白样结构域对于酶活性不是必需的。此外，与更大的 I 类和 II 类酶的结构相比，对这种植物 IV 类几丁质酶的晶体结构的比较表明，IV 类几丁质酶已经进化以适应较短的底物。
