A simple assay for 3-deoxy-d-manno-octulosonate cytidylyltransferase and its use as a pathway screen

Abstract

This paper describes the adaptation of a simple colorimetric assay for inorganic pyrophosphate to the enzyme 3-deoxy-d-manno-octulosonate cytidylyltransferase(CMP-KDO synthetase, KdsB, E.C. 2.7.7.38), a key enzyme in the biosynthesis of lipopolysaccharide (LPS) in Gram-negative organisms. This assay is particularly useful because it can be combined with the malachite green assay for inorganic phosphateto form an assay system capable of determining inorganic phosphate and inorganic pyrophosphate in the same solution (the MG/EK assay). This assay system has the potential for simultaneous screening of the 3-deoxy-d-manno-octulosonate (KDO) biosynthesis pathway. We test this potential using two enzymes, KdsB and KdsC, involved in the biosynthesis and utilization of the key bacterial 8-carbon sugar, KDO.

Analysis of the Arabinose-5-Phosphate Isomerase of Bacteroides fragilis Provides Insight into Regulation of Single-Domain Arabinose Phosphate Isomerases

“…The Eikonogen assay (19,20), which allows for the direct correlation of the concentration of CMP-Kdo with the amount of inorganic pyrophosphate produced, was used to determine the concentration of inorganic pyrophosphate produced. In this assay, the CMP-Kdo solution (50 l) was treated with 2.5% ammonium molybdate (50 l), 0.5 M ␤-mercaptoethanol (50 l,) and Eikonogen reagent (20 l, 0.125 g sodium sulfite, 7.325 g sodium metabisulfite, and 0.125 g 1-amino-2-naphthol-4-sulfonic acid in 50 ml hot deionized water).…”

“…The mixture was incubated at room temperature (ϳ21°C) for 30 min, and the absorbance was read at 540 nm. A malachite green assay, as previously described, was used to correct for inorganic phosphate concentrations (20). Absorbances were compared to a standard curve prepared using a serial dilution of a standard aqueous solution of Na 2 P 2 O 7 .…”

“…This limitation was circumvented by performing API assays using CMP-Kdo that was generated in situ using CTP, Kdo, and CMP- Kdo synthetase (KdsB) from E. coli. This process generated high micromolar concentrations of CMP-Kdo, as determined using the Eikonogen assay (20). The addition of CMP-Kdo-containing mixtures to Q5LIW1 enzymatic assays caused a substantial decrease in the activity of Q5LIW1 (K i of 1.91 Ϯ 0.48 M) (Fig.…”

Comparative analysis and validation of the malachite green assay for the high throughput biochemical characterization of terpene synthases

“…We adapted the malachite green (MG) assay as a fast, accurate, and inexpensive alternative to existing methods for measuring kinetics of phosphate-producing enzymes. The MG assay has been used for a number of years as a simple colorimetric method for the determination of inorganic phosphate [18–25] as well as for kinetic analysis and evaluation of enzymes involved in primary metabolism [26–31] . On one occasion, it has been used for estimating optimal conditions for the activity of a prenyl diphosphate synthase [32] .…”

A triple‐targeting inhibitory activity of Rose Bengal on polysaccharide biosynthesis of Burkholderia pseudomallei

“…D-Glycero-α-D-manno-heptose-1-phosphate guanylyltransferase (HddC), D-glycero-β-D-manno-heptose-1-phosphate adenylyltransferase (HldC), and 3-deoxy-D-manno-oct-2-ulosonic acid cytidylyltransferase (KdsB) are sugar nucleotidyltransferases involved in GDP-D-glycero-α-D-manno-heptose, ADP-L-glycero-β-Dmanno-heptose, and CMP-3-deoxy-D-manno-oct-2-ulosonic acid biosynthesis pathways, respectively. [3][4][5] Unfortunately, the KdsB gene deletion experiment has not been reported. However, a deletion mutant of the hldE gene from Escherichia coli working at the ADP-L-glycero-β-D-manno-heptose biosynthesis pathway, producing a bifunctional enzyme with activities of HldA and HldC, displayed increased vulnerability toward some antibiotics.…”