CHARACTERIZATION OF THE TYPE-I DEHYDROQUINASE FROM SALMONELLA-TYPHI

被引:34
作者
MOORE, JD
HAWKINS, AR
CHARLES, IG
DEKA, R
COGGINS, JR
COOPER, A
KELLY, SM
PRICE, NC
机构
[1] UNIV GLASGOW, DEPT BIOCHEM, GLASGOW G12 8QQ, SCOTLAND
[2] UNIV NEWCASTLE UPON TYNE, DEPT BIOCHEM & GENET, NEWCASTLE UPON TYNE NE2 4HH, ENGLAND
[3] WELLCOME RES LABS, DEPT MOLEC BIOL, BECKENHAM BR3 3BS, KENT, ENGLAND
[4] UNIV STIRLING, DEPT BIOL & MOLEC SCI, STIRLING FK9 4LA, SCOTLAND
[5] UNIV GLASGOW, DEPT CHEM, GLASGOW G12 8QQ, SCOTLAND
关键词
D O I
10.1042/bj2950277
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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. typhienzyme [Boys, Fawcett. Sawver, 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.
引用
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页码:277 / 285
页数:9
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