Premium
CdpNPT, an N‐Prenyltransferase from Aspergillus fumigatus : Overproduction, Purification and Biochemical Characterisation
Author(s) -
Yin WenBing,
Ruan HanLi,
Westrich Lucia,
Grundmann Alexander,
Li ShuMing
Publication year - 2007
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200700079
Subject(s) - prenyltransferase , prenylation , aspergillus fumigatus , chemistry , biochemistry , indole test , enzyme , stereochemistry , enzyme kinetics , tryptophan , divalent , heterologous expression , substrate (aquarium) , amino acid , active site , biology , gene , organic chemistry , recombinant dna , microbiology and biotechnology , ecology
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 tryptophan‐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 μ 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 Ca 2+ 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 CC bonds.