Crystal structure of a monocotyledon (maize ZMGlu1) β-glucosidase and a model of its complex with p-nitrophenyl β-D-thioglucoside
Crystal structure of a monocotyledon (maize ZMGlu1) β-glucosidase and a model of its complex with p-nitrophenyl β-D-thioglucoside
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DOI：
10.1042/0264-6021:3540037
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发表时间：
2001-02-15
影响因子：
4.1
通讯作者：
Esen, A
中科院分区：
文献类型：
--
作者：
Czjzek, M;Cicek, M;Esen, A
玉米α-葡萄糖苷酶同工酶ZMGlu1和ZMGlu2能降解丰富的天然底物DIMBOAGlc(2-O-β--D-glucopyranosyl-4-hydroxy-7-methoxy-1，4-benzoxazin-3-one)，，其苷元DIMBOA(2，4-羟基-7-甲氧基-1，4-苯并恶嗪-3-酮)是保护幼苗和幼苗免受食草动物和其他害虫侵害的主要防御化学物质。这两种同工酶对DIMBOAGlc的降解动力学相似，但彼此不同，对其他底物的专一性不同，也不同于高粱同系物。为了深入了解这两种玉米同工酶及其高粱同源物之间底物(即苷元)专一性的机制，在大肠杆菌中生产ZMGlu1，纯化、结晶并用X射线结晶学在2.5埃分辨率下解析其结构。此外，我们还对ZMGlu1与非水解性抑制剂对-硝基苯基-β-D-硫代葡萄糖苷的络合物进行了结晶，并根据局部电子密度提出了活性中心内的抑制剂分子模型。该缓蚀剂位于一个狭缝状的活性中心，其芳香族苷元通过与Trp-378的堆积作用而被保持。虽然葡萄糖部分非还原末端的一些原子可以根据电子密度进行模拟，但大多数抑制剂原子是高度无序的。这归因于酶需要适应两种不同的物种，即底物的基态和扭曲的构象，以进行催化。
The maize a-glucosidase isoenzymes ZMGlu1 and ZMGlu2 hydrolyse the abundant natural substrate DIMBOAGlc (2-O-beta -D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one), whose aglycone DIMBOA (2,4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) is the major defence chemical protecting seedlings and young plant parts against herbivores and other pests. The two isoenzymes hydrolyse DIMBOAGlc with similar kinetics but differ from each other and their sorghum homologues with respect to specificity towards other substrates. To gain insights into the mechanism of substrate (i.e. aglycone) specificity between the two maize isoenzymes and their sorghum homologues, ZMGlu1 was produced in Escherichia coli, purified, crystallized and its structure solved at 2.5 Angstrom resolution by X-ray crystallography. In addition, the complex of ZMGlu1 with the non-hydrolysable inhibitor p-nitrophenyl beta -D-thioglucoside was crystallized and, based on the partial electron density, a model for the inhibitor molecule within the active site is proposed. The inhibitor is located in a slot-like active site where its aromatic aglycone is held by stacking interactions with Trp-378. Whereas some of the atoms on the non-reducing end of the glucose moiety can be modelled on the basis of the electron density, most of the inhibitor atoms are highly disordered. This is attributed to the requirement of the enzyme to accommodate two different species, namely the substrate in its ground state and in its distorted conformation, for catalysis.