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H G Floss

Publications and source records attributed to H G Floss.

At least 37 records · Page 2Linked to original sources
PubMed1998

The granaticin biosynthetic gene cluster of Streptomyces violaceoruber Tü22: sequence analysis and expression in a heterologous host.

BACKGROUND: The granaticins are members of the benzoisochromanequinone class of aromatic polyketides, the best known member of which is actinorhodin made by Streptomyces coelicolor A3(2). Genetic analysis of this class of compounds has played a major role in the development of hypotheses about the way in which aromatic polyketide synthases (PKSs) control product structure. Although the granaticin nascent polyketide is identical to that of actinorhodin, post-PKS steps involve different pyran-ring stereochemistry and glycosylation. Comparison of the complete gene clusters for the two metabolites is therefore of great interest. RESULTS: The entire granaticin gene cluster (the gra cluster) from Streptomyces violaceoruber T-22 was cloned on either of two overlapping cosmids and expressed in the heterologous host, Streptomyces coelicolor A3(2), strain CH999. Chemical analysis of the recombinant strains demonstrated production of granaticin, granaticin B, dihydrogranaticin and dihydrogranaticin B, which are the four known metabolites of S. violaceoruber. Analysis of the complete 39,250 base pair sequence of the insert of one of the cosmids, pOJ466-22-24, revealed 37 complete open reading frames (ORFs), 15 of which resemble ORFs from the act (actinorhodin) gene cluster of S. coelicolor A3(2). Among the rest, nine resemble ORFs potentially involved in deoxysugar metabolism from Streptomyces spp. and other bacteria, and six resemble regulatory ORFs. CONCLUSIONS: On the basis of these resemblances, putative functional assignments of the products of most of the newly discovered ORFs were made, including those of genes involved in the PKS and tailoring steps in the biosynthesis of the granaticin aglycone, steps in the deoxy sugar pathway, and putative regulatory and export functions.

Amino Acid Sequence↗
PubMed1998

Nuclear magnetic resonance and biosynthetic studies of neoantimycin and structure elucidation of isoneoantimycin, a minor metabolite related to neoantimycin.

In preparation for biosynthetic studies on the 3,4-dihydroxy-2, 6-dimethyl-5-phenylvaleric acid portion of neoantimycin (1), the 1H and 13C NMR signals of 1 were assigned unambiguously by means of 2D correlation spectroscopy and NOE experiments. The previously undetermined absolute stereochemistry at C-15 and C-16 was deduced as (S) and (S). The structure of isoneoantimycin (2) was also elucidated. The methyl groups of methionine and propionate were incorporated stereospecifically into C-13 and C-12 of 1, respectively, and the configuration of the methyl group of methionine is inverted in the process. The results also suggest the intervention of phenylpyruvate as an actual precursor.

Anti-Bacterial Agents↗
PubMed1997

Stereochemistry of the in vitro and in vivo methylation of DNA by (R)- and (S)-N-[2H1,3H]methyl-N-nitrosourea and (R)- and (S)-N-nitroso-N-[2H1,3H]methyl-N-methylamine.

Reaction of DNA with the carcinogens N-methyl-N-nitrosourea and N-nitroso-N,N-dimethylamine produces several methylated species including the premutagenic O6-methylguanine. The mechanism of methylation is believed to be through a methanediazonium ion. We have studied the mechanism of methylation of DNA by these carcinogens by analyzing the stereochemistry of the methyl transfer. DNA was methylated in vitro by (R)- and (S)-N-[2H1,3H]methyl-N-nitrosourea and in vivo by (R)- and (S)-N-[2H1,3H]methyl-N-methyl-N-nitrosamine and (R)- and (S)-N-[2H1,3H]methyl-N-nitrosourea. 7-Methylguanine, 3-methyladenine, O6-methylguanine, and the methylated phosphate backbone were isolated. The methyl groups were converted into acetic acid, and the stereochemistry was analyzed. The identity of the nucleophile did not influence the stereochemistry of the methylation reaction. It was found that the methyl group was transferred with an average of 73% inversion and 27% retention of configuration. The most likely mechanism for the retention of configuration is through multiple methylation events in which nucleophiles which initially react with the methanediazonium ion react as electrophiles with DNA.

Alkylating Agents↗
PubMed1996

Mechanism of taxadiene synthase, a diterpene cyclase that catalyzes the first step of taxol biosynthesis in Pacific yew.

The first committed step in the formation of taxol has been shown to involve the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene. The formation of this endocyclic diterpene olefin isomer as the precursor of taxol was unexpected, since the exocyclic isomer, taxa-4(20),11(12)-diene, had been predicted as the initial product of the taxol pathway on the basis of metabolite co-occurrence. [1-2H2,20-2H3] and [20-2H3]geranylgeranyl diphosphates were employed as substrates with the partially purified taxadiene synthase from Pacific yew (Taxus brevifolia) stems to examine the possibility of a preliminary cyclization to taxa-4(20),11(12)-diene followed by isomerization to the more stable endocyclic double bond isomer. GLC-MS analysis of the derived taxa-4(5),11(12)-diene, via selected ion monitoring of the parent ion and the P-15 and C-ring fragment ions, compared to those of unlabeled standard, showed the olefin product to possess a deuterium enrichment essentially identical to that of the acyclic precursor, thus ruling out the putative isomerization step. With [4-2H2]geranylgeranyl diphosphate as substrate, similar product analysis established the enzymatically derived taxa-4(5),11(12)-diene to contain only one deuterium atom, consistent with direct formation from a taxenyl cation by deprotonation at C5. (+/-)-Casbene, (+/-)-verticillene, and (+/-)-taxa-4(20),11(12)-diene were tested as possible olefinic intermediates in taxa-4(5),11(12)-diene formation by a series of inhibition, trapping, and direct conversion experiments; no evidence was obtained that these exogenous olefins could serve as intermediates of the cyclization reaction. However, GLC-MS analysis of the taxadiene product derived by enzymatic cyclization of [1-3H]geranylgeranyl diphosphate in 2H2O indicated little incorporation of deuterium from the medium and suggested a rapid internal proton transfer in a tightly bound olefinic intermediate. Analysis of the enzymatic product generated from [10-2H1]geranylgeranyl diphosphate confirmed the intramolecular hydrogen transfer from C11 of a verticillyl intermediate to the C-ring of taxa-4(5),11(12)-diene. From these results, a stereochemical mechanism is proposed for the taxadiene synthase reaction involving the initial cyclization of geranylgeranyl diphosphate to a transient verticillyl cation intermediate, with transfer of the C11 alpha-proton to C7 to initiate transannular B/C-ring closure to the taxenyl cation, followed by deprotonation at C5 to yield the taxa-4(5),11(12)-diene product directly.

Isomerases↗
PubMed1996

Synthesis of [6"-3H]-, (6"-2H)- and (2-2H)-maltotriose.

To prepare labeled precursors for biosynthetic studies, methods for the specific introduction of tritium and deuterium into the reducing and the terminal glucose unit of maltotriose were developed. Thus [6"-3H]- and (6"-2H)-maltotriose (17) and (18) were prepared via selective methoxytritylation, deprotection and subsequent modified Pfitzner-Moffatt oxidation, followed by reduction with sodium borotritiide or sodium borodeuteride, respectively. A simple two step procedure utilizing the Lobry de Bruyn/van Ekenstein transformation gave (2-2H)maltotriose (20).

Carbohydrate Sequence↗
PubMed1996

Studies on the biosynthesis of thiostrepton: 4-(1-hydroxyethyl)quinoline-2-carboxylate as a free intermediate on the pathway to the quinaldic acid moiety.

Specifically 13C-labeled quinoline-2-carboxylate derivatives were synthesized from quinoline and used to study the biosynthesis of thiostrepton in a strain of Streptomyces laurentii. 13C NMR analysis of thiostrepton recovered after feeding methyl (RS)-[11-13C]-4-(1-hydroxyethyl)quinoline-2-carboxylate or methyl [11-13C]-4-acetylquinoline-2-carboxylate showed conclusively that these compounds are specifically and efficiently incorporated into thiostrepton. Both compounds were also detected in cultures of the producing organism by isotope dilution analysis. The significance of the relative endogenous concentrations of the two compounds and of the relative extent of the incorporation of exogenously added labeled material into thiostrepton are discussed in terms of the biosynthetic pathway linking tryptophan and 4-(1-hydroxyethyl)quinoline-2-carboxylate in S. laurentii. A highly specific enzyme activity was detected in cell-free extracts of S. laurentii that was capable of adenylating (12S)-4-(1-hydroxyethyl)quinoline-2-carboxylic acid. Partial purification of the enzyme was achieved. The enzyme was found to be specific for the enantiomer of the substrate which has the same absolute configuration as found in the natural antibiotic structure. The presence of one specific enzyme catalysing the adenylation process in S. laurentii was shown by photoaffinity labeling with [alpha-32P]-8-azido-ATP and subsequent SDS PAGE analysis of the labeled products. The native molecular weight of the active enzyme, determined by gel permeation chromatography, was found to be approximately 47 kDa, compared with a denatured weight of 50 kDa estimated for the photoaffinity-labeled protein. The enzyme is thus probably monomeric.

Affinity Labels↗
PubMed1995

The thiostrepton-resistance-encoding gene in Streptomyces laurentii is located within a cluster of ribosomal protein operons.

A common approach to identify and clone biosynthetic gene from an antibiotic-producing streptomycete is to clone the resistance gene for the antibiotic of interest and then use that gene to clone DNA that is linked to it. As a first step toward cloning the genes responsible for the biosynthesis of thiostrepton (Th) in Streptomyces laurentii (Sl), the Th resistance-encoding gene (tsnR) was cloned as a 1.5-kb BamHI-PvuII fragment in Escherichia coli (Ec), and shown to confer Th resistance when introduced into S. lividans TK24. The tsnR-containing DNA fragment was used as a probe to isolate clones from cosmid libraries of DNA in the Ec cosmid vector SuperCos, and pOJ446 (an Ec/streptomycete) cosmid vector. Sequence and genetic analysis of the DNA flanking the tsnR indicates that the Sl tsnR is not closely linked to biosynthetic genes. Instead it is located within a cluster of ribosomal protein operons.

Amino Acid Sequence↗
PubMed1995

Identification of Streptomyces violaceoruber Tü22 genes involved in the biosynthesis of granaticin.

A 50 kb region of DNA from Streptomyces violaceoruber Tü22, containing genes encoding proteins involved in the biosynthesis of granaticin, was isolated. The DNA sequence of a 7.3 kb fragment from this region, located approximately 10 kb from the genes that encode the polyketide synthetase responsible for formation of the benzoisochromane quinone skeleton, revealed five open reading frames (ORF1-ORF5). The deduced amino acid sequence of GraE, encoded by ORF2, shows 60.8% identity (75.2% similarity) to a dTDP-glucose dehydratase (StrE) from Streptomyces griseus. Cultures of Escherichia coli containing plasmids with ORF2, on a 2.1 kb BamHI fragment, were able to catalyze the formation of dTDP-4-keto-6-deoxy-D-glucose from dTDP-glucose at 5 times the rate of control cultures, confirming that ORF2 encodes a dTDP-glucose dehydratase. The amino acid sequence encoded by ORF3 (GraD) is 51.4% identical (69.9% similar) to that of StrD, a dTDP-glucose synthase from Streptomyces griseus. The amino acid sequence encoded by ORF4 shares similarities with proteins that confer resistance to tetracycline and methylenomycin, and is suggested to be involved in transporting granaticin out of the cells by an active efflux mechanism.

Amino Acid Sequence↗
PubMed1995

Thiopeptides.

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Amino Acid Sequence↗
PubMed1995

Three new omega-cycloheptyl fatty acids from Alicyclobacillus cycloheptanicus and their biosynthetic interrelationships.

Three minor omega-cycloheptyl fatty acids from Alicyclobacillus cycloheptanicus have been identified as omega-cycloheptylnonanoate, -decanoate, and -alpha-hydroxytridecanoate. The biosynthetic interrelations of these cyclic fatty acids have been studied. 13C-Labeled omega-cycloheptylundecanoate and -alpha-hydroxyundecanoate were converted to omega-cycloheptyldecanoate, which is one carbon shorter in length. alpha-Hydroxylation of omega-cycloheptylundecanate was also observed, but not chain extension to omega-cycloheptyltridecanoate.

Bacillus↗
PubMed1994

Overexpression of the thiostrepton-resistance gene from Streptomyces azureus in Escherichia coli and characterization of recognition sites of the 23S rRNA A1067 2'-methyltransferase in the guanosine triphosphatase center of 23S ribosomal RNA.

The thiostrepton-resistance gene encoding the 23S rRNA A1067 methyltransferase from Streptomyces azureus has been overexpressed in Escherichia coli using a T7-RNA-polymerase-dependent expression vector. The protein was efficiently expressed at levels up to 20% of total soluble protein and purified to near homogeneity. Kinetic parameters for S-adenosyl-L-methionine (Km = 0.1 mM) and an RNA fragment containing nucleotides 1029-1122 of the 23S ribosomal RNA from E. coli (Km = 0.001 mM) were determined. S-Adenosyl-L-homocysteine showed competitive product inhibition (Ki = 0.013 mM). Binding of either thiostrepton or protein L11 inhibited methylation. RNA sequence variants of the RNA fragment with mutations in nucleotides 1051-1108 were tested as substrates for the methylase. The experimental data indicate that methylation is dependent on the secondary structure of the hairpin including nucleotide A1067 and the exact sequence U(1066)-A(1067)-G(1068)-A(1069)-A(1070) of the single strand.

Base Sequence↗
PubMed1994

Biosynthetic studies on the origin of the cyclohexanecarboxylic acid moiety of ansatrienin A and omega-cyclohexyl fatty acids.

Feeding experiments with [2,6,10,10-2H4]chorismate in Streptomyces collinus (ansatrienin A) and Alicyclobacillus acidocaldarius (omega-cyclohexyl fatty acids), and inhibitor experiments with glyphosate in the latter organism, have shown that the biosynthesis of cyclohexanecarboxylic acid branches off from the shikimate pathway at a point prior to enolpyruvylshikimate 3-phosphate, either at shikimate or shikimate 3-phosphate.

Bacillaceae↗
PubMed1992

Structural determination of alginic acid and the effects of calcium binding as determined by high-field n.m.r.

The nature of the solution conformations of the alginic acid components D-mannuronan (poly-ManA) and L-guluronan (poly-GulA) from Azotobacter vinelandii were investigated by both one- and two-dimensional n.m.r. methods. Unequivocal proton assignments for both polymers as well as their constituent monomer units were made based on chemical-shift theory, coupling constant analysis, and nuclear Overhauser enhancement measurements. These data were used to investigate the interactions of poly-GulA and poly-ManA with Ca2+ ion in aqueous medium. Based on relative crosspeak integrals measured in two-dimensional phase-sensitive NOESY spectra of free and calcium-bound polymer, a model for calcium binding is proposed.

Alginates↗
PubMed1992

Biosynthesis of pyrrolnitrin. Incorporation of 13C, 15N double-labelled D- and L-tryptophan.

Experiments on the incorporation of D- and L-[alanine-3-13C,2-15N]tryptophan into the antibiotic pyrrolnitrin in Pseudomonas aureofaciens confirmed earlier conclusions about the conversion of L-tryptophan into pyrrolnitrin. They also demonstrated that a fraction of the D isomer is incorporated without breakage of the 15N-carbon bond, consistent with the operation of a second pathway from D-tryptophan to pyrrolnitrin. Cell-free experiments confirmed the conversion of 3-(o-aminophenyl)pyrrole into aminopyrrolnitrin but failed to detect enzymatic oxidation of the latter to pyrrolnitrin.

Alanine↗