Garcia-Casado G, Collada C, Allona I, Casado R, Pacios L F, Aragoncillo C, Gomez L
Departamento de Biotecnologia, E.T.S. Ingenieros de Montes, Universidad Politecnica de Madrid, E-28040 Madrid, Spain.
Glycobiology. 1998 Oct;8(10):1021-8. doi: 10.1093/glycob/8.10.1021.
Despite the intensive research on plant chitinases, largely bolstered by their antifungal properties, little is known at present about the structure-activity relationships of these enzymes. Here we report the identification of essential active site residues in endochitinase Ch3, a class I enzyme abundant in chestnut seeds. Knowledge-based protein modeling as well as structural and sequence comparisons were performed to identify potential catalytic residues. Different mutated proteins were then generated by site-directed mutagenesis, expressed in Escherichia coli , and characterized for their chitinolytic activity. Glu124 and Glu146, the only carboxylic residues properly located into the active site cleft to participate in catalysis, were both mutated to Gln and Asp. Our results suggest that Glu124 functions as the general acid catalyst whereas Glu146 is likely to act as a general base. Other mutations involving three highly conserved active site residues, Gln173, Thr175, and Asn254, also impaired the chitinolytic activity of Ch3. The effects of these variants on the fungus Trichoderma viride revealed that catalysis is not necessary for antifungal activity. Similarly to its homologous nonenzymatic polypeptides hevein and stinging nettle lectin, the N-terminal chitin-binding domain of Ch3 appears to interfere itself with hyphal growth.
尽管对植物几丁质酶进行了深入研究，主要是因为它们的抗真菌特性，但目前对这些酶的构效关系了解甚少。在此，我们报告了内切几丁质酶Ch3中必需活性位点残基的鉴定，Ch3是一种在板栗种子中大量存在的I类酶。通过基于知识的蛋白质建模以及结构和序列比较来鉴定潜在的催化残基。然后通过定点诱变产生不同的突变蛋白，在大肠杆菌中表达，并对其几丁质分解活性进行表征。活性位点裂隙中唯一适当定位以参与催化的羧基残基Glu124和Glu146均突变为Gln和Asp。我们的结果表明，Glu124作为一般酸催化剂起作用，而Glu146可能作为一般碱起作用。涉及三个高度保守的活性位点残基Gln173、Thr175和Asn254的其他突变也损害了Ch3的几丁质分解活性。这些变体对真菌绿色木霉的影响表明，催化对于抗真菌活性不是必需的。与其同源非酶多肽橡胶素和荨麻凝集素类似，Ch3的N端几丁质结合结构域似乎自身会干扰菌丝生长。
