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-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 isomerization in GDP-D-α-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 Km ~ 0.31 ± 0.06 mM-1 and coverts it to D-glycero-D-α-manno-heptose-7-phosphate with catalytic efficiency (kcat/Km) ~ 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% α-helix, ~ 19% β-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.

• Authors: Sumita Karan, Bhanu Pratap, Shiv Pratap Yadav, Fnu Ashish, Ajay K Saxena
• Journal: Scientific reports (Sci Rep) Vol. 10 Issue 1 Pg. 20813 (11 30 2020) ISSN: 2045-2322 [Electronic] England
• PMID: 33257730 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Bacterial Proteins
• Lipopolysaccharides
• Sugar Phosphates
• sedoheptulose 7-phosphate
• Aldose-Ketose Isomerases
• phosphoheptose isomerase

Topics

• Aldose-Ketose Isomerases (chemistry, metabolism)
• Bacterial Proteins (metabolism)
• Circular Dichroism
• Lipopolysaccharides (biosynthesis)
• Models, Molecular
• Mycobacterium tuberculosis (enzymology)
• Sugar Phosphates (metabolism)