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Structural and functional characterization of M. tuberculosis sedoheptulose- 7-phosphate isomerase, a critical enzyme involved in lipopolysaccharide biosynthetic pathway.

ABSTRACT: M. tuberculosis GmhA enzyme catalyzes the isomerization of D-sedoheptulose 7-phosphate into D-glycero-D-?-manno-heptose-7-phosphate in GDP-D-glycero-?-D-manno-heptose biosynthetic pathway. The D-glycero-?-D-manno-heptose is a major constituent of lipopolysaccharide and contributes to virulence and antibiotic resistance to mycobacteria. In current study, we have performed the structural and biochemical analysis of M. tuberculosis GmhA, the first enzyme involved in D-sedoheptulose 7-phosphate isomerization in GDP-D-?-D-heptose biosynthetic pathway. The MtbGmhA enzyme exits as tetramer and small angle X-ray scattering analysis also yielded tetrameric envelope in solution. The MtbGmhA enzyme binds to D-sedoheptulose 7-phosphate with K_m?~?0.31?±?0.06 mM^-1 and coverts it to D-glycero-D-?-manno-heptose-7-phosphate with catalytic efficiency (k_cat/K_m)?~?1.45 mM^-1 s^-1. The residues involved in D-sedoheptulose 7-phosphate and Zn²⁺ binding were identified using modeled MtbGmhA?+?D-sedoheptulose 7-phosphate?+?Zn²⁺ structure. To understand the role in catalysis, six site directed mutants of MtbGmhA were generated, which showed significant decrease in catalytic activity. The circular dichroism analysis showed?~?46% ?-helix, ~?19% ?-sheet and?~?35% random coil structures of MtbGmhA enzyme and melting temperature?~?53.5 °C. Small angle X-ray scattering analysis showed the tetrameric envelope, which fitted well with modeled MtbGmhA tetramer in closed conformation. The MtbGmhA dynamics involved in D-sedoheptulose 7-phosphate and Zn²⁺ binding was identified using dynamics simulation and showed enhanced stability in presence of these ligands. Our biochemical data and structural knowledge have provided insight into mechanism of action of MtbGmhA enzyme, which can be targeted for novel antibiotics development against M. tuberculosis.

SUBMITTER: Karan S

PROVIDER: S-EPMC7705670 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Structural and functional characterization of M. tuberculosis sedoheptulose- 7-phosphate isomerase, a critical enzyme involved in lipopolysaccharide biosynthetic pathway.

Karan Sumita S Pratap Bhanu B Yadav Shiv Pratap SP Ashish Fnu F Saxena Ajay K AK

Scientific reports 20201130 1

M. tuberculosis GmhA enzyme catalyzes the isomerization of D-sedoheptulose 7-phosphate into D-glycero-D-α-manno-heptose-7-phosphate in GDP-D-glycero-α-D-manno-heptose biosynthetic pathway. The D-glycero-α-D-manno-heptose is a major constituent of lipopolysaccharide and contributes to virulence and antibiotic resistance to mycobacteria. In current study, we have performed the structural and biochemical analysis of M. tuberculosis GmhA, the first enzyme involved in D-sedoheptulose 7-phosphate isom ...[more]

PMID: 33257730

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Project description:The complex formed between the enzyme triose phosphate isomerase (EC 5.3.1.1.), from rabbit and chicken muscle, and its substrate dihydroxyacetone phosphate was studied by 31P n.m.r. Two other enzyme-ligant complexes examined were those formed by glycerol 3-phosphate (a substrate analogue) and by 2-phosphoglycollate (potential transition-state analogue). Separate resonances were observed in the 31P n.m.r. spectrum for free and bound 2-phosphoglycollate, and this sets an upper limit to the rate constant for dissociation of the enzyme-inhibitor complex; the linewidth of the resonance assigned to the bound inhibitor provided further kinetic information. The position of this resonance did not vary with pH but remained close to that of the fully ionized form of the free 2-phosphoglycollate. It is the fully ionized form of this ligand that binds to the enzyme. The proton uptake that accompanies binding shows protonation of a group on the enzyme. On the basis of chemical and crystallographic information [Hartman (1971) Biochemistry 10, 146--154; Miller & Waley (1971) Biochem. J. 123, 163--170; De la Mare, Coulson, Knowles, Priddle & Offord )1972) Biochem. J. 129, 321--331; Phillips, Rivers, Sternberg, Thornton & Wilson (1977) Biochem. Soc. Trans. 5, 642--647] this group is believed to be glutamate-165. On the other hand, the position of the resonance of D-glycerol 3 phosphate (sn-glycerol 1-phosphate) in the enzyme-ligand complex changes with pH, and both monoanion and dianon of the ligand bind, although dianion binds better. The substrate, dihydroxyacetone phosphate, behaves essentially like glycerol 3-phosphate. The experiments with dihydroxy-acetone phosphate and triose phosphate isomerase have to be carried out at 1 degree C because at 37 degrees C there is conversion into methyl glyoxal and orthophosphate. The mechanismof the enzymic reaction and the reasons for rate-enhancement are considered, and aspects of the pH-dependence are discussed in an Appendix.

Dataset Information

Structural and functional characterization of M. tuberculosis sedoheptulose- 7-phosphate isomerase, a critical enzyme involved in lipopolysaccharide biosynthetic pathway.

Publications

Structural and functional characterization of M. tuberculosis sedoheptulose- 7-phosphate isomerase, a critical enzyme involved in lipopolysaccharide biosynthetic pathway.

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