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Crystallization and initial crystallographic analysis of phosphomannomutase/phosphoglucomutase from Pseudomonas
C A Regni1, P A Tipton, L J Beamer
1Biochemistry Department, University of Missouri, Columbia 65211, USA.
Abstract:
The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) catalyzes the conversion of mannose 6-phosphate to mannose 1-phosphate in the second step of the alginate biosynthetic pathway of Pseudomonas aeruginosa. PMM/PGM has been crystallized by hanging-drop vapor diffusion in space group P2(1)2(1)2(1). Crystals diffract to 1.75 A resolution on a synchrotron X-ray source under cryo-cooling conditions. PMM/PGM substituted with selenomethionine has been purified and crystallizes isomorphously with the native enzyme. Structure determination by MAD phasing is under way. Because of its role in alginate biosynthesis, PMM/PGM is a potential target for therapeutic inhibitors to combat P. aeruginosa infections.
Insights
The phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme is crucial for Pseudomonas aeruginosa alginate production. Crystallization and structural analysis are underway, targeting PMM/PGM for new therapeutic inhibitors against infections.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- Pseudomonas aeruginosa requires alginate for biofilm formation and virulence.
- The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) is essential in the alginate biosynthesis pathway.
- PMM/PGM catalyzes the conversion of mannose 6-phosphate to mannose 1-phosphate.
Purpose of the Study:
- To determine the crystal structure of PMM/PGM from P. aeruginosa.
- To facilitate the development of targeted therapeutic inhibitors.
- To understand the structural basis of alginate biosynthesis in P. aeruginosa.
Main Methods:
- Crystallization of PMM/PGM using hanging-drop vapor diffusion.
- X-ray diffraction data collection at 1.75 A resolution using synchrotron radiation.
- Purification and crystallization of selenomethionine-substituted PMM/PGM for MAD phasing.
Main Results:
- PMM/PGM was successfully crystallized in space group P2(1)2(1)2(1).
- High-resolution diffraction data were obtained under cryo-cooling conditions.
- Isomorphous crystallization of selenomethionine-substituted enzyme achieved.
Conclusions:
- PMM/PGM is a viable target for structure-based drug design.
- Structural insights into PMM/PGM could lead to novel anti-pseudomonal therapies.
- Inhibiting PMM/PGM may disrupt alginate production and combat P. aeruginosa infections.