귀리 Type Ⅰ β-Glucosidase multimer의 구조와 기능
Structure and function of oat type Ⅰ β-glucosidase multimer
작물학 귀리 β-Glucosidase multimer;
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β-Glucosidase (E.C. 220.127.116.11) that catalyzes the hydrolysis of the β-glycosidic bonds of aryl- and alkyl-β-glucoside and the cellobiose is widely distributed in all living organisms, and the aglycone of the glucosides controls substrate specificity of each enzyme. Plant β-glucosidases play roles in a variety of fundamental biological processes, e.g. a defense mechanism against pathogens and herbivores, phytohormone activation and cell wall degradation in the endosperm during germination by releasing aglycones. Thus, various physiological functions of the enzymes depend on their specific in vivo substrates. An oat β-glucosidase hydrolyzes β-glycosidic bond at the C26 of avenacosides to yield the 26-desglucoavenocosides, which are involved in defending the oat seedlings against fungal infections. When the cell is attacked by fungi, the cell wall and membranes are damaged and the enzyme in plastid and the avenacosides in vacuole are mixed together to hydrolize avenacoside. In oat, β-glucosidase forms characteristic, three dimensionally radiated arrangements of fibrils. We elucidated the fibrillar assembly of oat type I β-glucosidase by means of cryo-electron microscopy. It was assembled by linear stacking of hollow trimeric units and the resulting fibril had a long central tunnel connecting to the outer medium via regularly distributed side fenestrations. However, the packing of the monomers within the multimers was different from that within the hexamer. The length of two stacked trimers revealed within the long multimer was 9.0 nm, versus that of 12.7 nm in the free hexamers. This results indicates that the packing within the assemblies were much tighter when the trimers assembled into multimers and less tight when the assemblies were chopped up into hexamers. Tighter packing into the multimers also involves slight rotation between the trimers, as well as an increase in the outer dimensions of the basic unit. When the hexameric ring is projected down to the longitudinal axis, it results in pure six-fold symmetry in the projection, indicating that two trimers are rotated by 60 degrees to each other upon stacking. On the other hand, the multimers digress from this canonical arrangement, and the trimeric blocks were rotated by 38 degrees to each other. Tighter packing of subunits also results in slight change in the diameter of the multimer. Thus, the diameter changes from 10.8 nm in hexameric ring, which is uniform around the circumference of the assembly, to 11.4 nm in one direction and 9.0 nm perpendicular to it. Although the amino acid sequence of As-Glu 2 is highly homologous to that of As-Glu 1 except for their C-terminal portions, As-Glu 1 was assembled to form long fibrillar homomultimers, but As-Glu 2 formed mainly a dimer. A deletion mutant of the C-terminus of the As-glu 1 indicated that C-terminus sequence may not affected multimer formation but make the multimer stacks loose. Chimeric β-glucosidase were constructed by cDNA domain swapping to find a critical site for multimer formation. The chimeric β-glucosidase that was composed of 1-480 amino acids of As-glu 2 and 481-519 amino acids of As-glu 1 was assembled to multimer, but the other chimeric β-glucosidase that was composed of 1-499 amino acids of As-glu 2 and 500-519 amino acids of As-glu 1 formed mainly a dimer. Thus, the crucial binding sites for multimer formation were identified to be located in M480-N499. The difference in amino acids sequence between As-Glu 1 and As-Glu 2 in this region was K497 amino acids of As-Glu 1 and E496 amino acids of As-Glu 2 as the same position of K497 amino acids of As-Glu 1. E496K mutant of As-Glu 2 was assembled to multimer but K497E mutant of As-Glu 1 didn't affect the ability of multimer formation. Consequently, E496 amino acid of As-Glu 2 was critical site for the multimer formation. Enzyme kinetic studies of oat β-glucosidase multimers revealed distinct kinetic characteristics. Larger multimers had higher affinity to substrates than the smaller multimers, and the largest multimers hydrolysed the β-glycosidic linkage at a lower rate than the hexamer. Together with the experiment of active site inactivation, these indicated that the active site was located on the inner of fibrillar multimers. The results suggests that the oligomerisation of the oat type I β-glucosidase enhances the affinity of the enzyme to the avenacosides, against the competing substrates. As many different kinds of saponins are present in the plant cells, it is thus critical that the oat β-glucosidase specifically hydrolyzes only the avenacosides, the saponin integrated into the defense mechanism against fungal infections. In summary, the quarternary organization and intermediate resolution 3D structure of the oat *-glucosidase were revealed by the means of cryo-electron microscopy and image processing. The multimerization of the enzyme promotes formation of a long central channel with narrow openings at the sides. The crucial sites of multimer formation were revealed by cDNA swapping and site-directed mutagenesis. E496 amino acid of As-Glu 2 was critical site for the multimer formation. The structural characterizations were completed with the enzyme kinetics and chemical modification, also aimed to different oligomerization states. Enzyme kinetics study indicates that the enzymatic active site is localized inside the multimeric structures. Such configuration is likely to endow the enzyme with high affinity to substrates.
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