Enzyme study: PMM/PGM and its substrates

Nearly all Gram-negative bacteria possess lipopolysaccharide (LPS), an immunogenic molecule depicted in Figure 1. This structure is highly conserved across various microbes and plays a pivotal role in triggering the innate immune response in host organisms. When purified LPS is injected into experimental animals, it can induce endotoxic shock, often resulting in fatal outcomes.

Figure 1 Structure of LPS

The precursor of LPS is glucose-1-phosphate, which is formed when the enzyme phosphoglucomutase (PGM) rearranges glucose-6-phosphate. Conversely, PGM can also convert ample supplies of glucose-1-phosphate, sourced from glycogen, into glucose-6-phosphate—a fundamental molecule for both glycolysis and the pentose phosphate pathways. PGM has emerged as a potential target for antibacterial therapy due to its pivotal role; inhibiting PGM could disrupt LPS production and impede bacterial utilization of glucose-1-phosphate as an energy source.

In the Gram-negative bacterium P. aeruginosa, the enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) is essential for the biosynthesis of LPS and alginate, another molecule implicated in bacterial virulence. Phosphomannomutase (PMM) functionality rearranges mannose-6-phosphate into mannose-1-phosphate, a precursor of alginate, believed to encapsulate bacterial cells, shielding them from phagocytosis. PMM/PGM comprises four domains, three of which share a similar three-dimensional fold.

A team of researchers isolated the PMM/PGM enzyme through gel filtration and subjected it to analysis via SDS-PAGE. In Figure 2, lane 1 displays a standard protein ladder, lane 2 depicts the PMM/PGM sample before filtration, and lanes 3 to 5 depict sequential purifications of the enzyme."

Figure 2 Results of SDS PAGE analysis

Researchers also assessed the K$m$‍ and V$max$‍ values of the PMM/PGM enzyme for its substrates by Lineweaver-Burke analysis. Results are shown in Table 1.

Table 1 Kinetics of the bifunctional PMM/PGM enzyme