Gene cluster for streptomycin biosynthesis in<i>Streptomyces griseus</i>: nucleotide sequence of three genes and analysis of transcriptional activity

Distler, Jürgen; Ebert, A; Mansouri, Kambiz; Pissowotzki, Klaus; Stockmann, Michael; Piepersberg, Wolfgang

doi:10.1093/nar/15.19.8041

Cited by 159 publications

(96 citation statements)

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“…Orf4.1 is thus @A and not @C or @D as suggested previously ; the correct identification of @C and @D will be described later. The glucose-l-phosphate thymidylyltransferase of strain LT2 shows 34 % identity to that of Streptomyces greceus (strD of [15]; personal communication, W. Pipersberg). The SDSPAGE estimated subunit mass for rfbA protein of 31 kDa agrees well with the value of 32453 kDa, calculated from the amino acid composition deduced from the nucleotide sequence of the orf6.1 gene.…”

Section: Reaction Directionmentioning

confidence: 99%

Purification, characterization and HPLC assay of Salmonella glucose‐1‐phosphate thymidylyltransferase from the cloned rfbA gene

Lindquist

Kaiser

Reeves

et al. 1993

European Journal of Biochemistry

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We here report on the purification and characterization of glucose-1-phosphate thymidylyltransferase, the first of four enzymes commited to biosynthesis of dTDP-L-rhamnose from Salmonella enterica strain LT2. The purification was greatly facilitated by the cloning of the IjhA gene encoding this enzyme. Pure enzyme was obtained by 109-fold enrichment in three chromatography steps.The glucose-1 -phosphate thymidylyltransferase catalyzes a reversible bimolecular group transfer reaction and kinetic measurements indicate that it acts by a 'ping-pong' mechanism. The K , values for dTTP and a-D-glucose 1-phosphate in the forward reaction are 0.020 mM and 0.11 mM, respectively. In the reverse reaction the K , values for dTDP-D-glucose and diphosphate are 0.083 mM and 0.15 mM, respectively. The enzyme also accepts UTP and UDP-D-glucose and a-D-glucosamine 1-phosphate is accepted equally as well as a-D-glucose 1-phosphate.The NH,-terminal sequence of glucose-1 -phosphate thymidylyltransferase agrees with the sequence predicted from the nucleotide sequence of the 0lf6.l gene of the rj& gene cluster. The SDS/ PAGE estimated subunit mass of 31 kDa agrees well with that calculated from the amino acid composition deduced from the nucleotide sequence of the 0rf6.l gene (32453 Da).Saccharides are important surface structures in eukaryotic and prokaryotic cells and involved in cell recognition phenomena. They occur as free saccharides and as glycoconjugates linked to proteins and lipids. One of the problems in studies of their activities is the difficulty in obtaining them in pure form in sufficient quantities. In recent years advances have been made in the chemical synthesis of saccharides [l]. However, saccharide synthesis is still a relatively complicated procedure, in particular when larger saccharides or large quantities are desirable. In the last few years we have been investigating the feasibility of in vitro enzymatic synthesis of the Salmonella enterica 0-antigen-specific oligosaccharide and the nucleotide sugar precursors using enzymes overexpressed by cloned genes [Z].The 0-antigen part of S. enterica sero-group B strains is a repeating tetramer oligosaccharide composed of abequose, mannose, rhamnose and galactose. This saccharide is assembled from appropriate nucleoside &phosphate monosaccharides. The enzymes which participate in the biosynthesis of S. enterica 0-antigen polysaccharide are encoded by genes Abbreviations. Buffer A, 50mM Tris/HCl pH7.6, 10mM MgCl, and 1 mM EDTA; buffer B, 20 mM Tris/HC pH 8.0, 1 mM MgC1, and 22% glycerol; buffer C, 50mM sodium phosphate pH 7.0, 1 M ammonium sulphate and 22% glycerol.Enzymes. Glucose-I-phosphate thymidylyltransferase (EC 2.7.7.24), inorganic pyrophoshatase (EC 3.6.1.1).which are located mostly in the rj8 gene cluster [3]. The rj& gene cluster has been cloned [4-61, and Escherichia coli K12 strains harboring plasmids containing different parts of the rj8 gene cluster of S. enten'ca LT2 was the source of the enzymes used for in vitro enzymatic synthesis of dTDP-Lrhamnose, ...

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Section: Reaction Directionmentioning

confidence: 99%

Purification, characterization and HPLC assay of Salmonella glucose‐1‐phosphate thymidylyltransferase from the cloned rfbA gene

Lindquist

Kaiser

Reeves

et al. 1993

European Journal of Biochemistry

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“…For the first three compounds, it has been reported that their biosynthesis is controlled by the action of specific regulatory genes located within the particular biosynthetic clusters (4,(7)(8)(9). Such regulatory genes have also been identified in other antibiotic clusters, such as bialaphos (10), streptomycin (11,12), and daunorubicin (13).…”

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confidence: 99%

A relA/spoT Homologous Gene from Streptomyces coelicolor A3(2) Controls Antibiotic Biosynthetic Genes

Martı́nez-Costa

Arias

Romero

et al. 1996

Journal of Biological Chemistry

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A 0.972-kilobase pair DNA fragment from Streptomyces lividans that induces the production of the bluepigmented antibiotic actinorhodine in S. lividans when cloned on a multicopy plasmid has led to the isolation of a 4-kilobase pair DNA fragment from Streptomyces coelicolor containing homologous sequence. Computer-assisted analysis of the DNA sequence revealed three putative open reading frames (ORFs), ORF1, ORF2, and ORF3. ORF2 extends beyond the sequenced DNA fragment, and its deduced product shares no similarities with any other known proteins in the data bases. ORF3 is also truncated, and its 41-amino acid C-terminal product is identical to the S. coelicolor adenine phosphoribosyltransferase. The 847-amino acid ORF1 protein, with a predicted molecular mass of 94.2 kDa, strongly resembled the relA and spoT gene products from Escherichia coli and the homologs from Vibrio sp. strain S14, Haemophilus influenzae, Streptococcus equisimilis H46A, and Mycoplasma genitalium. Unlike these proteins, the ORF1 amino acid sequence analysis revealed the presence of a putative ATP/GTP-binding domain. A mutant was generated by deleting most of the ORF1 gene that showed an actinorhodine-nonproducing phenotype, while undecylprodigiosin and the calcium-dependent antibiotic were unaffected. The mutant strain grew at a much lower rate than the wild-type strain, and spore formation was delayed. When the gene was propagated on a low copy number vector, not only was actinorhodine production restored, but actinorhodine and undecylprodigiosin production was enhanced in both the mutant and wild-type strains and morphological differentiation returned to wild-type characteristics. (p)ppGpp synthetase activity was not detected in purified ribosomes from the ORF1-deleted mutant, while it was restored by complementation of this strain.

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“…Also, the StrD proteins, which are encoded by both related str/sts gene clusters for the biosynthesis of Sm and OH-Sm in S. griseus and S. glaucescens, respectively, are significantly, but more distantly related proteins. The StrD proteins belong to the subfamily of dTDP-D-glucose synthases and they catalyse the first step in dTDP-L-rhamnose synthesis in both strains (Distler et al, 1987;Verseck, S. and Piepersberg, W., unpublished). However, with StrQ the highest degree of similarity was detected to the subfamily of CDP-glucose synthases which have so far only been described from Enterobacteria, Bacillus subtilis and Synechocystis sp.…”

Section: Similarity Of the Strq Protein To Proteins In The Data Basementioning

confidence: 99%

“…The genes/enzymes for the activation, modification, and transfer of NDP-hexose precursors are still incompletely known. The enzymes encoded by the genes strDELM, encoding a dTDP-D-glucose synthase (StrD), a dTDP-D-glucose 4,6-dehydratase (StrE), a dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase (StrM), and a dTDP-L-rhamnose synthase (StrL) occur in all (OH-)Sm-producing strains and seem to be involved in the formation of dTDP-L-dihydrostreptose (Distler et al, 1987;Pissowotzki et al, 1991;Verseck, S. and Piepersberg, W., unpublished). The gene for the dTDP-L-dihydrostreptose synthase catalysing the final step has still to be identified.…”

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The StrQ protein encoded in the gene cluster for 5′‐hydroxystreptomycin of Streptomyces glaucescens GLA.0 is a α‐ D‐glucose‐1‐phosphate cytidylyltransferase (CDP‐ D‐glucose synthase)

Beyer

Mayer

Piepersberg

1998

European Journal of Biochemistry

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The gene strQ was identified as the last gene of a putative transcription unit, strB1FGHPQ, located in the gene cluster for the production of 5′-hydroxy-streptomycin (OH-Sm) in Streptomyces glaucescens GLA.0. [In contrast, the corresponding operon in the str/sts-gene cluster of the Sm-producer Streptomyces griseus, strB1FGHIK, differs in the two distal genes; Mansouri, K. & Piepersberg, W. (1991) Mol. Gen. Genet. 228, 459Ϫ469.] The deduced StrQ protein exhibited similarities to members of the enzyme family of hexose-1-phosphate nucleotidylyltransferases (NDP-hexose synthases or pyrophosphorylases), with the strongest similarity to the subfamily of A-D-glucose-1-phosphate cytidylyltransferases (CDP-D-glucose synthases). The StrQ protein was heterologously expressed in Escherichia coli. The purified protein revealed an enzyme activity of that of a CDP-D-glucose synthase and a substrate specificity restricted to CTP and A-D-glucose 1-phosphate. The K m and V max values determined for CTP are 44 µM and 920 µM and for A-D-glucose 1-phosphate 195 µM and 1.06 mM, respectively. The CDP-D-glucose synthase activity was also detected in cells of S. glaucescens under the conditions of antibiotic production, but was absent from cells of the streptomycin producer S. griseus N2-3-11. Also, the genomes of several strains of S. griseus did not seem to possess strQ-related genes. In contrast, hybridisation experiments indicated that genes homologous to strQ were probably present in various other actinomycetes producing aminoglycosides. A possible function of the StrQ protein in the OH-Sm biosynthetic pathway of GLA.0 is discussed.

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Gene cluster for streptomycin biosynthesis inStreptomyces griseus: nucleotide sequence of three genes and analysis of transcriptional activity

Cited by 159 publications

References 26 publications

Purification, characterization and HPLC assay of Salmonella glucose‐1‐phosphate thymidylyltransferase from the cloned rfbA gene

Purification, characterization and HPLC assay of Salmonella glucose‐1‐phosphate thymidylyltransferase from the cloned rfbA gene

A relA/spoT Homologous Gene from Streptomyces coelicolor A3(2) Controls Antibiotic Biosynthetic Genes

The StrQ protein encoded in the gene cluster for 5′‐hydroxystreptomycin of Streptomyces glaucescens GLA.0 is a α‐ D‐glucose‐1‐phosphate cytidylyltransferase (CDP‐ D‐glucose synthase)

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