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J Distler

Publications and source records attributed to J Distler.

At least 19 recordsLinked to original sources
PubMed2000

Preparative synthesis of GDP-beta-L-fucose by recombinant enzymes from enterobacterial sources.

The 6-deoxyhexose L-fucose is an important and characteristic element in glycoconjugates of bacteria (e.g., lipopolysaccharides), plants (e.g., xyloglucans) and animals (e.g., glycolipids, glycoproteins, and oligosaccharides). The biosynthetic pathway of GDP-L-fucose starts with a dehydration of GDP-D-mannose catalyzed by GDP-D-mannose 4,6-dehydratase (Gmd) creating GDP-4-keto-6-deoxymannose which is subsequently converted by the GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase (WcaG; GDP-beta-L-fucose synthetase) to GDP-beta-L-fucose. Both biosynthetic genes gmd and wcaG were cloned from Escherichia coli K12 and the enzymes overexpressed under control of the T7 promoter in the expression vectors pET11a and pET16b, yielding both native and N-terminal His-tag fusion proteins, respectively. The activities of the Gmd and WcaG were analyzed. The enzymatic conversion from GDP-D-mannose to GDP-beta-L-fucose was optimized and the final product was purified. The formation of GDP-beta-L-fucose by the recombinant enzymes was verified by HPLC and NMR analyses. The His-tag fusion variants of the Gmd and WcaG proteins were purified to near homogeneity. The His-tag Gmd recombinant enzyme was inactive, whereas His-tag WcaG showed very similar enzymatic properties relative to the native GDP-beta-L-fucose synthetase. With the purified His-tag WcaG Km and Vmax values, respectively, of 40 microM and 23 nkat/mg protein for the substrate GDP-4-keto-6-deoxy-D-mannose and of 21 microM and 10 nkat/mg protein for the cosubstrate NADPH were obtained; a pH optimum of 7.5 was determined and the enzyme was stimulated to equal extend by the divalent cations Mg2+ and Ca2+. The Gmd enzyme showed a strong feedback inhibition by GDP-beta-L-fucose.

Carbohydrate Epimerases↗
PubMed1999

The AcbC protein from Actinoplanes species is a C7-cyclitol synthase related to 3-dehydroquinate synthases and is involved in the biosynthesis of the alpha-glucosidase inhibitor acarbose.

The putative biosynthetic gene cluster for the alpha-glucosidase inhibitor acarbose was identified in the producer Actinoplanes sp. 50/110 by cloning a DNA segment containing the conserved gene for dTDP-D-glucose 4,6-dehydratase, acbB. The two flanking genes were acbA (dTDP-D-glucose synthase) and acbC, encoding a protein with significant similarity to 3-dehydroquinate synthases (AroB proteins). The acbC gene was overexpressed heterologously in Streptomyces lividans 66, and the product was shown to be a C7-cyclitol synthase using sedo-heptulose 7-phosphate, but not ido-heptulose 7-phosphate, as its substrate. The cyclization product, 2-epi-5-epi-valiolone ((2S,3S,4S,5R)-5-(hydroxymethyl)cyclohexanon-2,3,4,5-tetrol), is a precursor of the valienamine moiety of acarbose. A possible five-step reaction mechanism is proposed for the cyclization reaction catalyzed by AcbC based on the recent analysis of the three-dimensional structure of a eukaryotic 3-dehydroquinate synthase domain (Carpenter, E. P., Hawkins, A. R., Frost, J. W., and Brown, K. A. (1998) Nature 394, 299-302).

Acarbose↗
PubMed1998

Cloning and transcriptional analysis of the rplKA-or f31-rplJL gene cluster of Streptomyces griseus.

A 5018-bp DNA fragment of the rpl/rpo BC gene cluster (here called the rif cluster) of Streptomyces griseus N2-3-11 was analysed by DNA sequencing and transcription studies. By sequence comparison of the deduced proteins, five genes and part of an open reading frame (orf) were identified. The genes encoding the ribosomal (r-) proteins L1 (rplA), L7/12 (rplJ), L10 (rplK) and L11 (rplL), a protein of known function (orf31), and the N-terminus of the beta subunit of RNA polymerase (rpoB), are organized in three operons, rplKA, rplJL and rpoB(C), and the monocistronic transcription unit orf31. The promoters of these transcription units, rplKp, orf31p, rplJp, and rpoBp, were identified and the growth-phase dependence of the transcription of these operons was analysed. Binding sites for the ribosomal proteins L1 and L10 were identified by sequence comparison, suggesting that the r-proteins RplA and RplJ are involved in feedback regulation of their respective operons by binding to specific RNA-binding sites present in both the mRNA and the 23S rRNA, as has been described for other bacteria. The analyses of the rpoBp promoter by means of promoter-probe plasmids suggested a possible attenuator-based regulatory mechanism for the transcription of the rpoB(C) operon.

Amino Acid Sequence↗
PubMed1997

Properties of C-terminal truncated derivatives of the activator, StrR, of the streptomycin biosynthesis in Streptomyces griseus.

The StrR protein is a DNA-binding protein activating the transcription of streptomycin biosynthesis of Streptomyces griseus N2-3-11 and Streptomyces glaucescens. A putative helix-turn-helix motif located between amino acid positions 207 and 227 of the StrR protein was identified as a prerequisite for its DNA-binding properties. Although, C-terminal truncated StrR proteins were able to interact with StrR-binding sites, they failed to activate transcription from the StrR-dependent promotor strB1p. Therefore, the C-terminal domain of StrR seemed to be necessary for its function as transcriptional activator.

Amino Acid Sequence↗
PubMed1997

Identification of stsC, the gene encoding the L-glutamine:scyllo-inosose aminotransferase from streptomycin-producing Streptomycetes.

Eight new genes, strO-stsABCDEFG, were identified by sequencing DNA in the gene cluster that encodes proteins for streptomycin production of Streptomyces griseus N2-3-11. The StsA (calculated molecular mass 43.5 kDa) and StsC (45.5 kDa) proteins - together with another gene product, StrS (39.8 kDa), encoded in another operon of the same gene cluster - show significant sequence identity and are members of a new class of pyridoxal-phosphate-dependent aminotransferases that have been observed mainly in the biosynthetic pathways for secondary metabolites. The aminotransferase activity was demonstrated for the first time by identification of the overproduced and purified StsC protein as the L-glutamine:scyllo-inosose aminotransferase, which catalyzes the first amino transfer in the biosynthesis of the streptidine subunit of streptomycin. The stsC and stsA genes each hybridized specifically to distinct fragments in the genomic DNA of most actinomycetes tested that produce diaminocyclitolaminoglycosides. In contrast, only stsC, but not stsA, hybridized to the DNA of Streptomyces hygroscopicus ssp. glebosus, which produces the monoaminocyclitol antibiotic bluensomycin; this suggests that both genes are specifically used in the first and second steps of the cyclitol transamination reactions. Sequence comparison studies performed with the deduced polypeptides of the genes adjacent to stsC suggest that the enzymes encoded by some of these genes [strO (putative phosphatase gene), stsB (putative oxidoreductase gene), and stsE (putative phosphotransferase gene)] also could be involved in (di-)aminocyclitol synthesis.

Amino Acid Sequence↗
PubMed1996

The str gene cluster for the biosynthesis of 5'-hydroxystreptomycin in Streptomyces glaucescens GLA.0 (ETH 22794): new operons and evidence for pathway-specific regulation by StrR.

Two divergently oriented operons, strXU and strVW, located within the gene cluster for 5'-hydroxystreptomycin (5'-OH-Sm) biosynthesis in Streptomyces glaucescens strain GAL.0 (ETH 22794), were analysed by DNA sequencing and transcription/regulation studies. Three genes, strU and strVW, are conserved in a similar arrangement but in a different location within the str/sts gene cluster of the Sm-producing strain S. griseus N2-3-11. The four putative products resemble NDP-4-ketohexose 3,5-epimerases (StrX, M(r) 20.2 kDa), NAD(P)-dependent oxidoreductases (StrU, 45.6 kDa), and ABC-transporters (StrV, 61.8 kDa; StrW, 63.4 kDa). These genes are apparently involved in the biosynthesis of 5'-OH-Sm because the promoters of both operons are activated in trans by the activator StrR of S. griseus N2-3-11, when cloned in S. lividans 66 TK23. A sequence motif resembling the consensus sequence GTTCGActG(N)11CagTcGAAc for binding of StrR was identified within the intergenic region of strX and strV. Specific binding of StrR to this site was demonstrated by gel retardation assays using purified His*Tag-StrR.

ATP-Binding Cassette Transporters↗
PubMed1995

The regulator of streptomycin gene expression, StrR, of Streptomyces griseus is a DNA binding activator protein with multiple recognition sites.

In Streptomyces griseus the expression of at least one streptomycin biosynthetic gene, strB1, is dependent on the pathway-specific activator protein StrR. We show here that StrR is a DNA-binding protein which specifically interacts with the strB1 promoter fragment. Footprinting experiments demonstrate that the StrR protein binds to an inverted repeat located upstream of the strB1 promoter. Further StrR-binding sites having the consensus sequence GTTCGActG(N)11CagTcGAAc were identified in the str-sts gene clusters of S. griseus and Streptomyces glaucescens by sequence comparison, gel retardation, and footprinting studies. The genetic and biochemical evidence strongly supports the model of the StrR protein activating the expression of streptomycin biosynthetic genes by interacting with multiple binding sites within the str-sts gene clusters of S. griseus and S. glaucescens.

Bacterial Proteins↗
PubMed1994

The nusG gene of Streptomyces griseus: cloning of the gene and analysis of the A-factor binding properties of the gene product.

The nusG gene of Streptomyces griseus was cloned and the nucleotide sequence determined. It encodes a protein with an identity of 76% to the reported receptor (VbrA) for VB-C, an autoregulatory factor in Streptomyces virginae. NusG protein was expressed in Escherichia coli. However, no binding activity for A-factor, an butyrolactone autoregulator in S. griseus very similar to VB-C, could be detected. The nusG gene of S. griseus does not seem to encode the A-factor-binding protein.

4-Butyrolactone↗
PubMed1992

Streptomycin biosynthesis and its regulation in Streptomycetes.

New insights into the gene orders, structures, evolution, and functions of streptomycin (Sm) biosynthetic genes (str) were gained via hybridization studies, determination of nucleotide sequences, and measurement of expression in the str gene clusters of Streptomyces griseus and S. glaucescens. Both str clusters showed considerable divergence in macro and micro structure. Genes putatively involved in pathways leading to the (dihydro-)streptose and N-methyl-L-glucosamine moieties of Sm were identified. Additional regulatory elements, such as gene strS and conserved TTA codons in the N-terminal sections of reading frames, are reported. Evidences for the involvement of physiological state, signal transduction, and activators in the control of Sm production are presented.

Bacterial Proteins↗
PubMed1988

A second streptomycin resistance gene from Streptomyces griseus codes for streptomycin-3"-phosphotransferase. Relationships between antibiotic and protein kinases.

Two genes, aphE and orf, coding for putative Mr 29,000 and Mr 31,000, proteins respectively, were identified in the nucleotide sequence of a 2.8 kbp DNA segment cloned from Streptomyces griseus N2-3-11. The aphE gene expressed streptomycin (SM) resistance and a SM phosphorylating enzyme in S. lividans strains. The two genes were found to be in opposite direction and seemed to share a common region of transcription termination. The aphE gene shows significant homology to the aph gene, encoding aminoglycoside 3'-phosphotransferase, APH(3'), from the neomycin-producing S. fradiae. The enzymatic specificity of the aphE gene product was identified to be SM 3"-phosphotransferase, APH(3"). The primary structure of the APH(3") protein is closely related to the members of the APH(3') family of enzymes. However, the APH(3") enzyme did not detectably phosphorylate neomycin or kanamycin. There is only low similarity of the protein to the APH(6) group of SM phosphotransferases. An evolutionary relationship between antibiotic and protein kinases is proposed.

Amino Acid Sequence↗
PubMed1987

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

Three streptomycin (SM) production genes from Streptomyces griseus clustered around aphD, the major resistance gene, have been sequenced: strB, coding for an aminocyclitol amidinotransferase, ORF5 (strR), a putative regulatory gene, and ORF1 (strD), possibly coding for a hexose nucleotidylating enzyme. Three promoters and at least five, partially overlapping, transcripts have been identified by S1 mapping and Northern blot experiments. aphD, the resistance gene, is transcribed from two promoters. One of them, located inside the strR gene, seems to be constitutive and the other is switched on later in the growth phase. The late transcripts cover the resistance gene (aphD) and a regulatory gene (strR) which controls the expression of strB.

Amino Acid Sequence↗
PubMed1987

Gene cluster for streptomycin biosynthesis in Streptomyces griseus: analysis of a central region including the major resistance gene.

A central segment of a cluster of biosynthetic genes for the antibiotic streptomycin cloned from Streptomyces griseus was analysed for open reading frames, as well as for transcriptional and translational activity. The nucleotide sequence revealed two significant open reading frames, ORF1 and APH(6), orientated in opposite directions and with a spacer of 885 bp between the start codons. The first, ORF1, had a coding capacity of 38 kDa. One open reading frame, APH(6), was identified as the major resistance gene coding for streptomycin 6-phosphotransferase, a protein of 307 amino acid residues and 33 kDa. Sequence determination of the first 14 N-terminal amino acid residues of the purified APH(6) enzyme protein was in agreement with the proposed primary structure. The possible identity of the presumed gene product of ORF1 with an in vitro translated protein (apparent molecular weight 41 kDa) is discussed. Comparison of the two APH(6) genes from S. griseus and the hydroxystreptomycin-producing S. glaucescens (cf. Vögtli and Hütter 1987) revealed 75% nucleotide sequence homology in the coding region and 74% conservation of the polypeptide sequence. Two protein domains which are highly conserved in other antibiotic and protein phosphotransferases were detected.

Amino Acid Sequence↗
PubMed1987

Rouleau formation of erythrocytes is influenced by thrombocytes.

Thrombocytes are activated by shearing and influence possibly the flow properties of blood. Erythrocytes resuspended in platelet poor plasma show a significantly increased deformability as well as reduced aggregability after addition of ADP as well as adrenaline in comparison with control. In platelet rich plasma the aggregation of erythrocytes becomes enhanced. This is discussed in respect of a release of macromolecules of platelets.

Arterial Occlusive Diseases↗
PubMed1981

Characterization of a membrane-associated receptor from bovine liver that binds phosphomannosyl residues of bovine testicular beta-galactosidase.

A receptor that binds the phosphomannosyl recognition marker of bovine testicular beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) was isolated from bovine liver membranes. The receptor was extracted from crude plasma membrane preparations with Triton X-100 and immunoprecipitated as a receptor--beta-galactosidase complex with anti-beta-galactosidase. The receptor was dissociated from the precipitate with mannose 6-phosphate, labeled with 125I, and purified on a beta-galactosidase-Sepharose 4B affinity matrix. A quantitative binding assay employing anti-beta-galactosidase and IgGsorb (formalin-fixed Staphylococcus aureus) was devised to study the binding of 125I-labeled receptor to beta-galactosidase. Maximal binding of receptor to enzyme occurred at pH values between 5.7 and 6.5. Divalent cations were not required for binding. The values of the dissociation constant obtained for beta-galactosidase varied between 200 nM observed with "lower uptake" forms and 20 nM for "higher uptake" forms of the enzyme. A number of phosphorylated monosaccharides were tested as inhibitors of binding of enzyme to receptor; mannose 6-phosphate and fructose 1-phosphate served as inhibitors and exhibited Ki values of 0.064 mM and 0.24 mM, respectively. The receptor has a subunit molecular weight of 215,000. Similar receptors were also demonstrated in Triton X-100 extracts of human skin fibroblasts, Chinese hamster ovary cells, and rat hepatocytes. These cell types are known to assimilate lysosomal enzymes containing covalently bound mannose 6-phosphate residues.

Animals↗
PubMed1980

Selective noncompetitive assimilation of bovine testicular beta-galactosidase and bovine liver beta-glucuronidase by generalized gangliosidosis fibroblasts.

Bovine liver beta-glucuronidase and testicular beta-galactosidase were assimilated by generalized gangliosidosis fibroblasts at respectively rates of 90 and 464 times the rate of assimilation of horseradish peroxidase. Assimilation of either of the two enzymes by the fibroblasts was saturable, suggesting the participation of receptor-mediated adsorptive endocytosis for internalization. The rate of assimilation of either enzyme was not affected by high levels of the other enzyme, suggesting that distinct receptors for each enzyme occur on the fibroblasts' cell surface. Furthermore, although assimilation of beta-galactosidase was inhibited by mannose, methyl mannosides, mannosyl alpha 1 leads to 2 mannose, and mannose-6-phosphate, these compounds did not detectably inhibit the assimilation of beta-glucuronidase. These results suggest that testicular beta-galactosidase was assimilated by the well-established phosphomannosyl recognition system. However, liver beta-glucuronidase was assimilated by a distinct, noncompeting, and as yet undefined, recognition system.

Animals↗