Study of PVCA and PVCB, two enzymes involved in making isonitrile-containing natural products in bacterial pathogens
Metadata[+] Show full item record
Isonitriles, with an unusual functionality, are very versatile intermediates widely involved in organic synthesis. Interestingly, a variety of isonitriles were encountered in both marine and terrestrial systems as the naturally occurring metabolites. Many of them were isolated as antibiotics; many of them were identified to play important roles in host-pathogen interactions; and a lot more remained uncharacterized. The chemical and functional diversity of these novel compounds may offer important synthetic implications and pharmaceutical potential. The gene clusters related to the biosynthesis of isonitrile-containing natural products have been identified in many bacteria. They commonly encode two novel proteins, designated PvcA and PvcB. Early functional studies of the proteins identified PvcA as an isonitrile synthase and PvcB as an oxygenase. But the in vitro activities of these proteins remained undetermined until recently. To demonstrate PvcB as an Fe[2+ superscript], [alpha]-ketoglutarate dependent oxygenase, and as a source of the diversity in isonitrile-containing metabolites, reactions catalyzed by the PvcB homologous proteins from Pseudomonas aeruginosa, Xenorhabdus nematophila and Erwinia amylovora were characterized and compared. Recombinant PvcB proteins were heterologously expressed and purified to homogeneity. Catalytic activities of three PvcB enzymes were evaluated in the presence of the synthesized substrate tyrosine isonitrile and the co-substrates Fe[2+ superscript] , [alpha]-ketoglutarate and O2 subscript]. UV-visible spectra revealed turnovers in all of the three reactions, as well as the difference between the XnPvcB reaction and the other two. Subsequent reaction product determination by [1 superscript]H-NMR spectroscopy and LCMS (liquid chromatography-mass spectrometry) demonstrated that XnPvcB catalyzed an oxidative decarboxylation reaction on tyrosine isonitrile, while the PaPvcB and EaPvcB reactions retained the carboxyl group. Steady-state and transient-state kinetics were performed to determine the catalytic efficiency and the mechanisms of the PvcB enzymes. And substrate docking and homology modeling were used to compare the active sites of the proteins. Our data suggested the various activities exhibited by the PvcB homologs from different pathways might result from the subtle differences of the proteins in the active sites. PvcA is a novel isonitrile synthase and is predicted to catalyze the conversion of the amino group into the isonitrile functionality. To determine its in vitro activity and gain a deeper understanding of the mechanism underlying the isonitrile group synthesis, PvcA proteins from Pseudomonas aeruginosa, Xenorhabdus nematophila and Aeromonas hydrophila were expressed and purified. Activity assays were performed with the putative substrates tyrosine and ribulose-5- phoshpate. However, reactions monitored by UV-visible spectroscopy and HPLC (high-performance liquid chromatography) did not show substrate transformation. No success was made in the reactions involving crude lysates instead of purified enzymes, either. To determine whether correct substrates were used, in vivo feeding and labeling experiments were performed in the biosynthetic pathway of isocyanovinylphenol to track the product backbone and the isonitrile carbon. Our data demonstrated tyrosine to be one of the substrates for the PvcA enzyme, and suggested the other substrate, which provides the isonitrile carbon might be a metabolite from the pentose phosphate pathway. In the meantime, by overexpressing XnPvcA in E. coli, we detected the PvcA product in both the cell lysate and the culture medium, and identified it as tyrosine isonitrile, further supporting the conclusion that tyrosine is the substrate providing the product backbone. In order to determine the isonitrile carbon precursor, a phosphorylated metabolite library, the central carbon metabolic pathways and the metabolite pool of E. coli cells were examined in search of this substrate, but the substrate was only found in the E. coli cell lysate. Fractionation of the lysate by filtration, dialysis, size-exclusion and ion exchange chromatography identified a macromolecule, like a metabolic enzyme, and a small compound, probably phosphorylated, were both required for PvcA activity.