Site-directed mutagenesis of putative active-site residues in squalene-hopene cyclase
Site-directed mutagenesis of putative active-site residues in squalene-hopene cyclase
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DOI：
10.1111/j.1432-1033.1996.0051r.x
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发表时间：
1996-11-01
期刊：
影响因子：
--
通讯作者：
Poralla, K
中科院分区：
文献类型：
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作者：
Feil, C;Sussmuth, R;Poralla, K
角鲨烯-藿烯环化酶 (SHC) 催化角鲨烯到藿烯的复杂多环化，类似于氧化角鲨烯到甾醇的环化。 SHC 的序列分析揭示了一个高度保守的富含天冬氨酸的基序 (DDTA)，与氧化角鲨烯环化酶的 DCTA 基序相当，它被认为是活性位点的一部分。为了确定该基序在角鲨烯环化中的重要性，将来自酸热脂环酸芽孢杆菌的SHC的DDTA基序中的保守残基Asp376和Asp377分别替换为谷氨酸、谷氨酰胺、甘氨酸和精氨酸。除[Glu376]SHC突变体外，所有其他取代均导致酶活性几乎或完全丧失。与野生型酶相比，[Glu376]SHC突变酶的比活性降低至10%，表观V-max显着降低，而表观K-m保持不变。 CD测量表明突变不影响二级结构。有人提出 Asp376 和 Asp377 对于催化至关重要，并且可以充当点电荷来稳定中间阳离子。此外，对于角鲨烯-霍烯环化酶，可以发现高含量的α-螺旋构象，为三萜环化酶提供了第一个结构信息。
Squalene-hopene cyclase (SHC) catalyzes the complex polycylization of squalene to hopene, similar to the cyclization of oxidosqualene to sterols. Sequence analysis of SHC revealed a highly conserved aspartate-rich motif (DDTA), comparable to the DCTA motif of oxidosqualene cyclases, which is supposed to be part of the active site. In order to determine the importance of the motif in squalene cyclization, the conserved residues Asp376 and Asp377 in the DDTA motif of SHC from Alicyclabacillus acidocaldarius were individually replaced by glutamate, glutamine, glycine, and arginine. With the exception of the [Glu376]SHC mutant, all other substitutions resulted in almost or complete loss of enzyme activity. Compared to that of the wild-type enzyme, the specific activity of the [Glu376]SHC mutant enzyme was reduced to 10%, accompanied by a significant decrease in the apparent V-max, whereas the apparent K-m remained unchanged. CD measurements indicated that mutations did not affect the secondary structure. It is proposed that Asp376 and Asp377 are crucial for catalysis and may act as point charges to stabilize intermediate cations. Moreover, for squalene-hopene cyclase, a high content of alpha-helical conformation could be found, providing the first structural information for a triterpene cyclase.