Moore J D, Hawkins A R, Charles I G, Deka R, Coggins J R, Cooper A, Kelly S M, Price N C
Department of Biochemistry and Genetics, University of Newcastle upon Tyne, U.K.
Biochem J. 1993 Oct 1;295 ( Pt 1)(Pt 1):277-85. doi: 10.1042/bj2950277.
The type I dehydroquinase from the human pathogen Salmonella typhi was overexpressed in an Escherichia coli host and purified to homogeneity. The S. typhi enzyme was characterized in terms of its kinetic parameters, important active-site residues, thermal stability and c.d. and fluorescence properties. In all important respects, the enzyme from S. typhi behaves in a very similar fashion to the well-characterized enzyme from E. coli, including the remarkable conformational stabilization observed on reduction of the substrate/product mixture by NaBH4. This gives confidence that the information from X-ray studies on the S. typhi enzyme [Boys, Fawcett, Sawyer, Moore, Charles, Hawkins, Deka, Kleanthous and Coggins (1992) J. Mol. Biol. 227, 352-355] can be applied to other type I dehydroquinases. Studies of the quenching of fluorescence of the S. typhi enzyme by succinimide show that NaBH4 reduction of the substrate/product imine complex involves a dramatic decrease in the flexibility of the enzyme, with only very minor changes in the overall secondary and tertiary structure.
人类病原体伤寒沙门氏菌的I型脱氢奎尼酸酶在大肠杆菌宿主中过表达并纯化至同质。对伤寒沙门氏菌的这种酶进行了动力学参数、重要活性位点残基、热稳定性以及圆二色性和荧光特性等方面的表征。在所有重要方面，伤寒沙门氏菌的这种酶与已得到充分表征的大肠杆菌的酶表现出非常相似的行为，包括在由硼氢化钠还原底物/产物混合物时观察到的显著构象稳定化。这让人相信，关于伤寒沙门氏菌酶的X射线研究[博伊斯、福西特、索耶、摩尔、查尔斯、霍金斯、德卡、克莱安托斯和科金斯（1992年）《分子生物学杂志》227卷，352 - 355页]所获得的信息可应用于其他I型脱氢奎尼酸酶。对琥珀酰亚胺淬灭伤寒沙门氏菌酶荧光的研究表明，硼氢化钠还原底物/产物亚胺复合物会使该酶的灵活性显著降低，而其整体二级和三级结构仅有非常微小的变化。
