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首页> 外文期刊>Applied Microbiology and Biotechnology >Functional dissection of the N-terminal sequence of Clostridium sp G0005 glucoamylase: identification of components critical for folding the catalytic domain and for constructing the active site structure
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Functional dissection of the N-terminal sequence of Clostridium sp G0005 glucoamylase: identification of components critical for folding the catalytic domain and for constructing the active site structure

机译：Clostridium SP G0005葡糖淀粉酶N-末端序列的功能解剖：鉴定折叠催化结构域并构建活性位点结构的组分

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摘要

Clostridium sp. G0005 glucoamylase (CGA) is composed of a beta-sandwich domain (BD), a linker, and a catalytic domain (CD). In the present study, CGA was expressed in Escherichia coli as inclusion bodies when the N-terminal region (39 amino acid residues) of the BD was truncated. To further elucidate the role of the N-terminal region of the BD, we constructed N-terminally truncated proteins (Delta 19, Delta 24, Delta 29, and Delta 34) and assessed their solubility and activity. Although all evaluated proteins were soluble, their hydrolytic activities toward maltotriose as a substrate varied: Delta 19 and Delta 24 were almost as active as CGA, but the activity of Delta 29 was substantially lower, and Delta 34 exhibited little hydrolytic activity. Subsequent truncation analysis of the N-terminal region sequence between residues 25 and 28 revealed that truncation of less than 26 residues did not affect CGA activity, whereas truncation of 26 or more residues resulted in a substantial loss of activity. Based on further site-directed mutagenesis and N-terminal sequence analysis, we concluded that the 26XaaXaaTrp28 sequence of CGA is important in exhibiting CGA activity. These results suggest that the N-terminal region of the BD in bacterial GAs may function not only in folding the protein into the correct structure but also in constructing a competent active site for catalyzing the hydrolytic reaction.
机译：Clostridium sp。 G0005葡糖淀粉酶（CGA）由β-夹心结构域（BD），接头和催化结构域（CD）组成。在本研究中，当Bd的N-末端区域（39个氨基酸残基）被截断时，CGA在大肠杆菌中表达。为了进一步阐明BD的N-末端区域的作用，我们构建了N-末端截短的蛋白质（Delta 19，Delta 24，Delta 29和Delta 34）并评估它们的溶解度和活性。尽管所有评估的蛋白质是可溶的，但它们作为底物的麦芽酮糖的水解活性变化：Delta 19和Delta 24几乎与CGA一样活性，但Delta 29的活性基本下降，Delta 34表现出很少的水解活性。残留物25和28之间的N-末端区域序列的后续截短分析显示，截短小于26个残基的截短不影响CGA活性，而26或更多残基的截短导致了大量的活性。基于进一步的现场定向诱变和N-末端序列分析，我们得出结论，26xAaxaATRP28 CGA序列在表现出CGA活性方面是重要的。这些结果表明，细菌气体中BD的N-末端区域不仅可以在将蛋白质折叠成正确的结构中，而且可以在构建用于催化水解反应的主动性的活性位点时起作用。

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