Characterization of the type I dehydroquinase from Salmonella typhi.
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.
Moore
J D
JD
Department of Biochemistry and Genetics, University of Newcastle upon Tyne, U.K.
Hawkins
A R
AR
Charles
I G
IG
Deka
R
R
Coggins
J R
JR
Cooper
A
A
Kelly
S M
SM
Price
N C
NC
D002942:Circular Dichroism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006639:Histidine / Q000378:metabolism; D006358:Hot Temperature; D006836:Hydro-Lyases / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000378:metabolism; D012485:Salmonella typhi / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013388:Succinimides / Q000494:pharmacology; D014364:Tryptophan
D006146:Guanidines; D011994:Recombinant Proteins; D013388:Succinimides; C032620:succinimide; D006639:Histidine; D014364:Tryptophan; D006836:Hydro-Lyases; C020000:3-dehydroquinate dehydratase; D019791:Guanidine; D004047:Diethyl Pyrocarbonate