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 properti...

Full description

Saved in:
Bibliographic Details
Published in:Biochemical journal 1993-10, Vol.295 (1), p.277-285
Main Authors: MOORE, J. D, HAWKINS, A. R, CHARLES, I. G, DEKA, R, COGGINS, J. R, COOPER, A, KELLY, S. M, PRICE, N. C
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Circular Dichroism
Diethyl Pyrocarbonate - pharmacology
Enzymes and enzyme inhibitors
Escherichia coli - genetics
Fundamental and applied biological sciences. Psychology
Guanidine
Guanidines - pharmacology
Histidine - metabolism
Hot Temperature
Hydro-Lyases - drug effects
Hydro-Lyases - genetics
Hydro-Lyases - isolation & purification
Hydro-Lyases - metabolism
Kinetics
Lyases
Oxidation-Reduction
Protein Conformation
Protein Denaturation
Recombinant Proteins - metabolism
Salmonella typhi - enzymology
Spectrometry, Fluorescence
Spectrophotometry
Succinimides - pharmacology
Tryptophan
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0264-6021
1470-8728
DOI:10.1042/bj2950277