A rapid assay for dihydropteroate synthase activity suitable for identification of inhibitors

Fernley, R, Iliades, P and Macreadie, I 2007, 'A rapid assay for dihydropteroate synthase activity suitable for identification of inhibitors', Analytical Biochemistry, vol. 360, no. 2, pp. 227-234.


Document type: Journal Article
Collection: Journal Articles

Title A rapid assay for dihydropteroate synthase activity suitable for identification of inhibitors
Author(s) Fernley, R
Iliades, P
Macreadie, I
Year 2007
Journal name Analytical Biochemistry
Volume number 360
Issue number 2
Start page 227
End page 234
Total pages 8
Publisher Academic Press
Abstract The enzymes 6-hydroxymethylpterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) catalyze sequential steps in folate biosynthesis. They are present in microorganisms but absent in mammals and therefore are especially suitable targets for antimicrobials. Sulfa drugs (sulfonamides and sulfones) currently are used as antimicrobials targeting DHPS, although resistance to these drugs is increasing. The most widely used assay that measures activity of these enzymes, to assess new inhibitors in vitro, is not amenable to automation. This article describes a simple, coupled, enzymatic spectrophotometric assay where the product of the DHPS reaction, dihydropteroate, is reduced to tetrahydropteroate by excess dihydrofolate reductase (DHFR) using the cofactor NADPH. The oxidation of NADPH is monitored at 340 nm. The activity of both HPPK and DHPS can be measured in this assay, and it has been used to measure kinetic parameters of wild-type and sulfa drug-resistant DHPS enzymes to demonstrate the utility of the assay. It is a sensitive and reproducible assay that can be readily automated and used in multiwell plates. This NADPH-coupled microplate photometric assay could be used for rapid screening of chemical libraries for novel inhibitors of folate biosynthesis as the first step in developing new antimicrobial drugs targeting the folate biosynthetic pathway.
Subject Analytical Chemistry not elsewhere classified
Keyword(s) Continuous assay
Dihydrofolate reductase
Dihydropteroate synthase
Folate
Hydroxymethyldihydropterin pyrophosphokinase
Spectrophotometric
Sulfa drugs
DOI - identifier 10.1016/j.ab.2006.10.036
Copyright notice Crown Copyright © 2006
ISSN 0003-2697
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