From a cosmid library of Streptomyces cyanogenus S136, DNA fragments encompassing approximately 35 kb of the presumed landomycin biosynthetic gene cluster were identified and sequenced, revealing 32 open reading frames most of which could be assigned through data base comparison.

A putative prenyltransferase gene, Afu3g12930, was identified in the genome sequence of Aspergillus fumigatus. EAL92290, encoded by Afu3g12930, consists of 472 aa, with a molecular mass of about 53 kDa. The coding sequence of Afu3g12930 was cloned in pQE60, and overexpressed in Escherichia coli. The soluble His 6 -fusion protein was purified to apparent homogeneity, and characterized biochemically. The enzyme was found to catalyse the prenylation of Trp at the C-7 position of the indole moiety, in the presence of dimethylallyl diphosphate (DMAPP); therefore, it functions as a 7-dimethylallyltryptophan synthase (7-DMATS). The structure of the enzymic product was elucidated by NMR and MS analysis. K m values were 67 mM for DMAPP, and 137 mM for L-Trp. Geranyl diphosphate was not accepted as prenyl donor, while Trp-containing dipeptides were found to be aromatic substrates of 7-DMATS. 7-DMATS did not need divalent metal ions for its enzymic reaction, although Ca 2+ enhanced the reaction velocity slightly. The enzyme is the second dimethylallyltryptophan synthase identified in A. fumigatus. Interestingly, it shares a sequence identity of only 31 % at the amino acid level with another known dimethylallyltryptophan synthase, FgaPT2, from the same fungus; FgaPT2 prenylates L-Trp at the C-4 position of the indole ring. Afu3g12930 belongs to a putative biosynthetic gene cluster consisting of eight genes. Orthologous clusters were also identified in the genome sequences of Neosartorya fischeri and Aspergillus terreus. The putative roles of the genes in the cluster are discussed.

Analysis of glycosyltransferase genes of the urdamycin biosynthetic gene cluster led to an unambiguous assignment of each glycosyltransferase to a certain biosynthetic saccharide attachment step.

Urdamycin A, the principal product of Streptomyces fradiae Tu $ 2717, is an angucycline-type antibiotic and anticancer agent containing C-glycosidically linked D-olivose. To extend knowledge of the biosynthesis of urdamycin A the authors have cloned further parts of the urdamycin biosynthetic gene cluster. Three new ORFs (urdK, urdJ and urdO) were identified on a 335 kb fragment, and seven new ORFs (urdL, urdM, urdJ2, urdZ1, urdGT2, urdG and urdH) on an 805 kb fragment. The deduced products of these genes show similarities to transporters (urdJ and urdJ2), regulatory genes (urdK), reductases (urdO), cyclases (urdL) and deoxysugar biosynthetic genes (urdG, urdH and urdZ1). The product of urdM shows striking sequence similarity to oxygenases (N-terminal sequence) as well as reductases (C-terminal sequence), and the deduced amino acid sequence of urdGT2 resembles those of glycosyltransferases. To determine the function of urdM and urdGT2, targeted gene inactivation experiments were performed. The resulting urdM deletion mutant strains accumulated predominantly rabelomycin, indicating that UrdM is involved in oxygenation at position 12b of urdamycin A. A mutant in which urdGT2 had been deleted produced urdamycin I, urdamycin J and urdamycin K instead of urdamycin A. Urdamycins I, J and K are tetracyclic angucyclinones lacking a C-C connected deoxysugar moiety. Therefore UrdGT2 must catalyse the earliest glycosyltransfer step in the urdamycin biosynthetic pathway, the C-glycosyltransfer of one NDP-D-olivose.

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 microM. 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 C--C bonds.