Cloning and Overexpression in Soluble Form of Functional Shikimate Kinase and 5-Enolpyruvylshikimate 3-Phosphate Synthase Enzymes from Mycobacterium tuberculosis

2001 ◽  
Vol 22 (3) ◽  
pp. 430-435 ◽  
Author(s):  
Jaim S. Oliveira ◽  
Clotilde A. Pinto ◽  
Luiz A. Basso ◽  
Diógenes S. Santos
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Soluble Form ◽  
Shikimate Kinase ◽  
Phosphate Synthase

scholarly journals A novel inhibitor of indole-3-glycerol phosphate synthase with activity against multidrug-resistant Mycobacterium tuberculosis

FEBS Journal ◽  
2008 ◽  
Vol 276 (1) ◽  
pp. 144-154 ◽  
Author(s):  
Hongbo Shen ◽  
Feifei Wang ◽  
Ying Zhang ◽  
Qiang Huang ◽  
Shengfeng Xu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Multidrug Resistant ◽  
Glycerol Phosphate ◽  
Phosphate Synthase

A High-Throughput Screening Assay for Simultaneous Selection of Inhibitors of Mycobacterium tuberculosis 1-Deoxy-D-Xylulose-5-Phosphate Synthase (Dxs) or 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (Dxr)

2011 ◽  
Vol 16 (3) ◽  
pp. 303-312 ◽  
Author(s):  
Vaishali Humnabadkar ◽  
Ramesh K. Jha ◽  
Nuzhat Ghatnekar ◽  
Sunita M. De Sousa
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Isothermal Calorimetry ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Enzyme Assays ◽  
High Concentration ◽  
High Throughput Screening Assay ◽  
Individual Enzyme ◽  
Phosphate Synthase

1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) is involved in the synthesis of isoprenoids by the methylerythritol phosphate pathway. Dxr is essential in Mycobacterium tuberculosis ( Mtu), absent in humans and amenable to structure-aided design. To further assess the druggability of the enzyme, the energetics of binding of fosmidomycin to Mtu Dxr was studied by isothermal calorimetry. Binding was enhanced by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) and driven by enthalpy (ΔH –10.2 kcal/mol, ΔS 1.1 cal mol−1K−1). This suggests the possibility of finding novel inhibitors that bind enthalpically, making Dxr an attractive target. The cost of the Dxr substrate, 1-deoxy-D-xylulose-5-phosphate, for high-throughput screening (HTS) is prohibitive. Hence, an HTS assay that couples Dxr to the upstream enzyme 1-deoxy-D-xylulose-5-phosphate synthase (Dxs), also a valid target, was developed. A high concentration of NADPH was used to bias it to detect Dxr inhibitors that bind like fosmidomycin. The assay Z′ was 0.75. It was equally sensitive to inhibitors of Dxs and Dxr, that is, fosmidomycin and fluropyruvate inhibited it with IC50s similar to that in the individual enzyme assays (79 vs 54 nM for fosmidomycin). To distinguish inhibitors of Dxs from Dxr, individual enzyme assays and a microplate thermofluor binding assay were developed. The assay simultaneously screens two targets and is cost-effective.

Purification and characterization of Mycobacterium tuberculosis indole-3-glycerol phosphate synthase

2006 ◽  
Vol 71 (S1) ◽  
pp. S38-S43 ◽  
Author(s):  
Yanping Yang ◽  
Min Zhang ◽  
Hongmei Zhang ◽  
Jianqiang Lei ◽  
Ruiliang Jin ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Glycerol Phosphate ◽  
Purification And Characterization ◽  
Phosphate Synthase

One-step purification of 5-enolpyruvylshikimate-3-phosphate synthase enzyme from Mycobacterium tuberculosis

2003 ◽  
Vol 28 (2) ◽  
pp. 287-292 ◽  
Author(s):  
Jaim S. Oliveira ◽  
Maria A. Mendes ◽  
Mário S. Palma ◽  
Luiz A. Basso ◽  
Diógenes S. Santos
Keyword(s):  
Mycobacterium Tuberculosis ◽  
One Step ◽  
Phosphate Synthase
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