A study of Rose Bengal against a 2-keto-3-deoxy-d-manno-octulosonate cytidylyltransferase as an antibiotic candidate

Abstract

Frequent occurrences of multi-drug resistance of pathogenic Gram-negative bacteria threaten human beings. The CMP-2-keto-3-deoxy- d - manno -octulosonic acid biosynthesis pathway is one of the new targets for antibiotic design. 2-Keto-3-deoxy- d - manno -octulosonate cytidylyltransferase (KdsB) is the key enzyme in this pathway. KdsB proteins from Burkholderia pseudomallei ( Bp ), B. thailandensis ( Bt ), Pseudomonas aeruginosa ( Pa ), and Chlamydia psittaci ( Cp ) have been assayed to find inhibitors. Interestingly, Rose Bengal (4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein) was turned out to be an inhibitor of three KdsBs ( Bp KdsB, Bt KdsB, and Pa KdsB) with promising IC 50 values and increased thermostability. The inhibitory enzyme kinetics of Rose Bengal revealed that it is competitive with 2-keto-3-deoxy- manno -octulosonic acid (KDO) but non-competitive against cytidine 5′-triphosphate (CTP). Induced-fit docking analysis of Pa KdsB revealed that Arg160 and Arg185 together with other interactions in the substrate binding site seemed to play an important role in binding with Rose Bengal. We suggest that Rose Bengal can be used as the scaffold to develop potential antibiotics.

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

“…Bengal against KDO. [18] The double reciprocal plots confirmed that BpHldC and BpKdsB are allosteric enzymes (Figures S3 and S4).…”

“…A malachite green assay, the photometric method, was carried out for the screening of chemical compounds (Table S1). [18] The reaction mechanism of the three enzymes is similar, except for their substrates.…”

“…HddC, HldC, and KdsB from B. pseudomallei (BpHddC, BpHldC, and BpKdsB) were obtained with the previously reported methods. [4,17,18] Three soluble proteins containing a noncleavable N-terminal His 6 tag were overexpressed and purified by Ni 2+ affinity chromatography. To further purify proteins, ion-exchange chromatography using a 5-ml Hi-Trap Q column was performed with the fractions from the affinity purifications.…”

“…The malachite green assay method [18] was used to study the steadystate kinetics of three proteins. For the kinetic studies, the reaction mixture (40 μl) consisted of Tris-HCl buffer (5 mM, pH 7.5), MgCl 2 (10 mM), IPP (0.04 unit) with BpHddC (0.025 mg/ml ), BpHldC (0.05 mg/ml), and BpKdsB (0.01 mg/ml), respectively.…”

A study of inhibitors of d-glycero-β-d-manno-heptose-1-phosphate adenylyltransferase from Burkholderia pseudomallei as a potential antibiotic target

“…In brief, affinity chromatography was done and ion-exchange chromatography has followed as the secondary purifications. KdsB proteins from B. pseudomallei , C. jejuni , Chlamydia psittaci , E. coli , Klebsiella aerogenes , N. meningitidis , Pseudomonas aeruginosa , Salmonella enterica , and Vibrio cholerae have been purified according to the previous method 17 . Purification was also carried out with the same process.…”