The sensor phosphodiesterase (PDE) NbdA from the opportunistic human pathogen Pseudomonas aeruginosa comprises three domains, an MHYT inner membrane domain, and the cytosolic tandem GGDEF-EAL domain. Previous experiments have demonstrated that the cytosolic domain of NbdA is capable of degrading the second messenger cyclic di-GMP (Li et al. 2013). This molecule regulates virulence and biofilm formation in P. aeruginosa (Römling et al. 2013). Recombinantly produced and purified full-length protein NbdA from E. coli BL21(DE3) did not show any phosphodiesterase activity. Interestingly, the full-length protein demonstrated activity when produced in E. coli Nissle 1917 in the presence of heme and iron ions.

The aim of this study is to elucidate the putative binding of heme to purified full-length NbdA and the dependency of PDE activity. The solubilization of the full-length protein from E. coli Nissle 1917 membrane fractions using polymer nanodiscs enabled spectroscopic characterization of heme binding to the protein. Absorption spectra of oxidized and reduced NbdA revealed characteristic Soret bands for heme proteins at 412 nm (oxidized) and 424 nm (reduced). Results from the pyridine-hemochrome method suggest a non-covalent binding of heme to NbdA.

In summary, in vivo supplementation of heme during heterologous production leads to PDE activity of NbdA. Combined with spectroscopic investigations, it is inferred that heme is specifically bound by NbdA. Further enzyme assays regarding NbdA's dependence on heme are currently underway.

References

Li et al. (2013) J. Bacteriol. 195 (16), 3531–3542.

Römling et al. (2013) Microbiol. Mol. Biol. Rev. 77 (1), 1–52.