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Unit 2: Lesson 1

Foundation 1: Biomolecules

The PMM/PGM enzyme and its substrates


Almost all Gram-negative bacteria release lipopolysaccharide (LPS), an immunogenic molecule shown in Figure 1. It is one of the most conserved microbial structures and is responsible for the activation of the innate immune response in host organisms. Injection of purified LPS into experimental animals can lead to endotoxic shock with lethal outcome.

Figure 1 Structure of LPS
A precursor of LPS is glucose-1-phosphate, which is created when the enzyme phosphoglucomutase (PGM) rearranges glucose-6-phosphate. Conversely, PGM can also convert large supplies of glucose-1-phosphate, obtained from glycogen, into glucose-6-phosphate, which is a beginning molecule for both the glycolysis and pentose phosphate pathways. PGM has therefore been a target for antibacterial therapy, since its inhibition could interfere with LPS production as well bacterial utilization of glucose-1-phosphate as an energy source.
The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from the Gram-negative bacterium P. aeruginosa is required for the biosynthesis of LPS as well as alginate, another molecule known to play a role in the virulence of the bacteria. The phosphomannomutase (PMM) functionality rearranges mannose-6-phosphate into mannose-1-phosphate, a precursor of alginate, which is believed to encapsulate bacterial cells and protect them from phagocytosis. PMM/PGM consists of four domains, three of which have a similar three-dimensional fold.
Researchers interested in studying a PMM/PGM blockade set up the following experiments.
Experiment 1
The Km and Vm values for substrates were determined from a Lineweaver-Burke analysis. The values are shown in Table 1.
SubstrateKstart subscript, m, end subscript (μM)Vstart subscript, m, a, x, end subscript (μM•molstart superscript, minus, 1, end superscript•mgstart superscript, minus, 1, end superscript)
Table 1: Kinetics of the bifunctional PGM/PMM enzyme.
Experiment 2
The PGM/PMM enzyme was extracted by gel filtration analyzed by SDS page (Figure 2). Lane 1 shows a standard protein ladder, lane 2 shows PGM/PMM prior to filtration, and lanes 3 to 5 show progressive purifications of the enzyme.

Figure 2
Sources: Ye, R. W., Zielinski, N. A., and Chakrabarty, A. M. (1994) Purification and characterization of phosphomannomutase/phosphoglucomutase from Pseudomonas aeruginosa involved in biosynthesis of both alginate and lipopolysaccharide, J Bacteriol 176, 4851-4857.
Where in the cell would LPS likely be found?
Choose 1 answer:
Choose 1 answer: