Purification and characterisation of the monodechloroaminopyrrolnitrin 3-halogenase PrnC from pyrrolnitrin biosynthesis
Zusammenfassung der Projektergebnisse
Monodechloroaminopyrrolnitrin 3-halogenase (PrnC) catalyses the third step in the biosynthesis of the antifungal antibiotic pyrrolnitrin. PrnC is a flavin-dependent halogenase and catalyses the regioselective incorporation of a chlorine atom into the 3-position of the pyrrole ring of pyrrolnitrin. PrnC accepts a free substrate, monodechloroaminopyrrolnitrin, which was produced by in vivo transformation of 7-chlorotryptophan by PrnB, the enzyme catalysing the second step in pyrrolnitrin biosynthesis. A new purification strategy was developed allowing the purification of larger amounts of PrnC which were required for biochemical characterisation and crystallisation. This purification procedure was based on the construction of a fusion of the PrnC gene to the gene of the maltose binding protein. It could be shown that purified PrnC can contain copper at a ratio of almost one mole of copper to one mole of enzyme, depending on the copper contend of the water used for preparation of the buffers used for its purification. However, it could be demonstrated that copper was not required for halogenating activity of PrnC. The kinetic parameters for the organic substrate MCD and chloride could be determined. These values are in the range of those published for other flavin-dependent halogenases. During the screening for crystallisation conditions for PrnC, first crystals of PrnC could be obtained, however, these crystals were much too small for X-ray analysis. A model of the three-dimensional structure of PrnC was constructed based on the known structure of the alkyl halogenase Cmls from chloramphenicol biosynthesis. As already known from the primary structure of PrnC, this halogenase does not seem to have the catalytically important lysine and glutamate residues. According to the model, PrnC has a serine residue at the position of the lysine residue of other halogenases. Thus variants in which the serine was exchanged against an alanine, lysine or threonine residue, were constructed and purified. Analysis of the kinetic data of these variants showed that they were essentially the same as for the wild type enzyme. Thus it can be concluded that the serine residue in PrnC does not substitute the catalytically important lysine residue of other flavin-dependent halogenases, leaving it unclear which amino acids in PrnC are important for halogenating activity. Using complementation assays, the substrate specificity will be further investigated in vivo and in vitro. For this purpose we are in the process of complementing a mutant of the pyoluteorin producer lacking a halogenase gene with prnC and we are also synthesising a peptide carrier protein tethered pyrrole carboxylic acid as a potential substrate.
Projektbezogene Publikationen (Auswahl)
- ESF-EMBO Symposium: Synthetic Biology of Antibiotic Production (Sant Feliu de Guixols, Spain (2011) "Purification of the MCAP 3-halogenase from pyrrolnitrin biosynthesis in P. fluorescens BL915"
A. Adam, K.-H. van Pée
- (2012) Biosynthesis of halogenated alkaloids. Alkaloids 71, 167-210
van Pée K.-H.
- (2012) Enzymatic chlorination and bromination. Methods Enzymol. 516, 237-257
van Pée, K.-H.
- (2012) Halogenating enzymes for selective halogenation reactions. Curr. Org. Chem. 16, 2583-2597
van Pée, K.-H.
- Jahrestagung 2013 der VAAM (Tübingen 2012) “Purification and characterisation of the flavin-dependent monodechloroaminopyrrolnitrin 3-halogenase from pyrrolnitrin biosynthesis”
A. Adam, K.-H. van Pée
- VAAM Workshop on the biology of bacteria producing natural products (Frankfurt (2013) “The phenylpyrrole halogenase PrnC from pyrrolnitrin biosynthesis – a new type of flavin-dependent halogenase?”
A. Adam, K.-H. van Pée
