The dnrM gene in Streptomyces peucetius contains a naturally occurring frameshift mutation that is suppressed by another locus outside of the daunorubicin-production gene cluster

Abstract

A 2.7 kb BamHl fragment of the daunorubicin biosynthetic cluster in Streptomyces peucetius ATCC 29050 was shown to contain two ORFs, dnrL and dnfM# whose deduced products exhibit a high sequence similarity to a number of glucose-l-phosphate thymidylyl transferases and TDP-D-glucose dehydratases, respectively. Although these genes were believed to be necessary for the synthesis of the deoxyaminosugar, daunosamine, a constituent of daunorubicin, the dnrM gene contains a frameshift in the DNA sequence that causes the premature termination of translation. A gene encoding another TDP-glucose 4#6=dehydratase, previously isolated from 5. peucetius, was identified by PCR amplification of genomic DNA. The presence of this gene explains why a dnrM::aphll mutation did not block daunorubicin production.

Cloning and characterization of the Streptomyces peucetius dnrQS genes encoding a daunosamine biosynthesis enzyme and a glycosyl transferase involved in daunorubicin biosynthesis

“…Proposed pathway for biosynthesis of DNR from propionyl-coenzyme A (CoA), malonyl-CoA, and glucose-1-OPO 3 . Open arrows indicate multiple steps between precursor and product, and the corresponding gene designations are shown: dpsA-G (12), dnrCD (26), dnrF (9), dnrP (26), dnrK (27), dnrL (11), and dnrJ (25). The sequence of enzymatic reactions following the formation of TDP-4-keto-6-deoxy-D-glucose in the daunosamine pathway is hypothetical.…”

“…A putative TDP-glucose synthetase gene, dnrL (11), has been identified in the DNR gene cluster (12,25,31) of Streptomyces peucetius ATCC 29050, and a TDP-glucose-4,6-dehydratase has been partially purified from this organism (37). Surprisingly, the dnrM gene, which is located adjacent to dnrL in the DNR gene cluster, encodes a 4,6-dehydratase homolog that appears to be nonfunctional because of a frameshift mutation which results in the synthesis of a truncated protein (11). These results led to the detection of another 4,6-dehydratase gene (11) located outside the DNR gene cluster which encodes the enzyme described by Thompson et al (37).…”

Cloning and characterization of the Streptomyces peucetius dnmZUV genes encoding three enzymes required for biosynthesis of the daunorubicin precursor thymidine diphospho-L-daunosamine

“…1), has been identified in the DNR gene cluster of the wild-type Streptomyces peucetius ATCC 29050 strain, and a thymidine-diphospho (TDP)-glucose-4,6-dehydratase for the formation of the 4-keto-6-deoxyhexulose nucleotide that is a key precursor in deoxyhexose biosynthesis has been purified (28). The gene encoding the putative TDP-glucose-4,6-dehydratase has been localized outside of the DNR gene cluster (8), while a TDP-glucose-4,6-dehydratase homolog, dnmM (formerly dnrM) (8), found adjacent to dnmL, is apparently nonfunctional due to a frameshift mutation. Inactivation of the dnmJ (formerly dnrJ) gene (17) indicates that it is required for daunosamine biosynthesis, and the similarity between DnmJ and AscC (29) suggests that DnmJ is likely to be involved in the addition of the C-3 amino group to a daunosamine precursor (30).…”

“…These data facilitate the construction of mutant strains that are disrupted in potential sugar genes and permit the assignment of reasonable functions to deduced gene products by sequence comparisons to proteins of known function, such as those for the biosynthesis of rhamnose (16) and the 3,6-dideoxyhexoses (14) that are found in the lipopolysaccharides of gram-negative bacteria. Thus a putative glucose-1-phosphate thymidylyltransferase gene, dnmL (formerly dnrL) (8), which presumably governs the first step of daunosamine biosynthesis (Fig. 1), has been identified in the DNR gene cluster of the wild-type Streptomyces peucetius ATCC 29050 strain, and a thymidine-diphospho (TDP)-glucose-4,6-dehydratase for the formation of the 4-keto-6-deoxyhexulose nucleotide that is a key precursor in deoxyhexose biosynthesis has been purified (28).…”

Cloning and insertional inactivation of Streptomyces argillaceus genes involved in the earliest steps of biosynthesis of the sugar moieties of the antitumor polyketide mithramycin

“…The highest homologies were with StrD (68.7% identity and 80.6% similarity) from the streptomycin pathway (13), GraD (54.1% identity and 71.9% similarity) from the granaticin pathway (1), SnoD (52.0% identity and 72.2% similarity) from Streptomyces nodosus with unknown function (GenBank accession no. A25110), and DnrL (68.3% identity and 79.7% similarity) from the daunorubicin pathway (6). The MtmE protein strongly resembled TDP-D-glucose 4,6-dehydratases from different antibiotic pathways: StrE (69.2% identity and 81.4% similarity) from the streptomycin pathway (13); TylA2 (62.8% identity and 73.8% similarity) from the tylosin pathway (10); GraE (60% identity and 75% similarity) from the granaticin pathway (1); and Gdh (65.6% identity and 76.2% similarity), a dehydratase from the erythromycin producer Saccharopolyspora erythraea, which has been shown not to be involved in antibiotic biosynthesis (7).…”