CdpNPT, an N-prenyltransferase from Aspergillus fumigatus:: Overproduction, purification and biochemical characterisation

A putative prenyltransferase gene, cdpNPT was identified in the genome sequence of Aspergillus fumigatus by a homology search by using known prenyltransferases and sequence analysis. CdpNPT consists of 440 amino acids and has a molecular mass of about 50 kDa. The coding sequence of cdpNPT was cloned in pQE60 and overexpressed in E. coli. The soluble His(6)-fusion CdpNPT was purified to near homogeneity and characterised biochemically. The enzyme showed broad substrate specificity towards aromatic substrates and was found to catalyse the prenylation of tryptophon-containing cyclic dipeptides at N1 of the indole moieties in the presence of dimethylallyl diphosphate (DMAPP); geranyl diphosphate was not accepted as prenyl donor. The structures of the enzymatic products were elucidated by NMR and MS analysis. The K-m value for DMAPP was determined to be 650 mu m. Due to substrate inhibition, K-m values could not be obtained for the aromatic substrates. CdpNPT does not need divalent metal ions for its enzymatic reaction, although Ca2+ enhances the reaction velocity by up to the threefold. CdpNPT is the first N-prenyltransferase that has been purified and characterised in a homogenous form after heterologous overproduction. Interestingly, it shows significant sequence similarity to other indole prenyltransferases that catalyse the formation of C-C bonds.