Purification and characterisation of the monodechloroaminopyrrolnitrin 3-halogenase PrnC from pyrrolnitrin biosynthesis

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Monodechloroaminopyrrolnitrin 3-halogenase (PrnC) catalyses the second halogenation step in pyrrolnitrin biosynthesis. Besides the tryptophan halogenases, PrnC has, until very recently, been the only flavin-dependent halogenase know to accept a free substrate. Interestingly, PrnC has no significant amino acid sequence homology to tryptophan halogenases. Thus, it is of great significance for the understanding of the reaction mechanism and the substrate specificity of flavin-dependent halogenases to characterise PrnC biochemically and to elucidate its 3-dimensional structure. For this purpose, monodechloroaminopyrrolnitrin, the substrate of PrnC, will be isolated and used for the detection of PrnC during purification to homogeneity. The kinetic data (Km, Vmax, kcat) for the organic and inorganic substrates will be determined. In addition, the substrate specificity and the regioselectivity of PrnC will be analysed in detail. Purified PrnC will allow the elucidation of the 3-D structure which in turn will allow the construction of mutants to clarify the role of specific amino acids in the reactions catalysed by PrnC and other flavin-dependent halogenases.