Search PubMed⌕ Search

Biomedical subjects

D H Sherman

Publications and source records attributed to D H Sherman.

At least 37 records · Page 2Linked to original sources
PubMed1999

Mitomycin resistance in Streptomyces lavendulae includes a novel drug-binding-protein-dependent export system.

Sequence analysis of Streptomyces lavendulae NRRL 2564 chromosomal DNA adjacent to the mitomycin resistance locus mrd (encoding a previously described mitomycin-binding protein [P. Sheldon, D. A. Johnson, P. R. August, H.-W. Liu, and D. H. Sherman, J. Bacteriol. 179:1796-1804, 1997]) revealed a putative mitomycin C (MC) transport gene (mct) encoding a hydrophobic polypeptide that has significant amino acid sequence similarity with several actinomycete antibiotic export proteins. Disruption of mct by insertional inactivation resulted in an S. lavendulae mutant strain that was considerably more sensitive to MC. Expression of mct in Escherichia coli conferred a fivefold increase in cellular resistance to MC, led to the synthesis of a membrane-associated protein, and correlated with reduced intracellular accumulation of the drug. Coexpression of mct and mrd in E. coli resulted in a 150-fold increase in resistance, as well as reduced intracellular accumulation of MC. Taken together, these data provide evidence that MRD and Mct function as components of a novel drug export system specific to the mitomycins.

Amino Acid Sequence↗
PubMed1998

A gene cluster for macrolide antibiotic biosynthesis in Streptomyces venezuelae: architecture of metabolic diversity.

In a survey of microbial systems capable of generating unusual metabolite structural variability, Streptomyces venezuelae ATCC 15439 is notable in its ability to produce two distinct groups of macrolide antibiotics. Methymycin and neomethymycin are derived from the 12-membered ring macrolactone 10-deoxymethynolide, whereas narbomycin and pikromycin are derived from the 14-membered ring macrolactone, narbonolide. This report describes the cloning and characterization of the biosynthetic gene cluster for these antibiotics. Central to the cluster is a polyketide synthase locus (pikA) that encodes a six-module system comprised of four multifunctional proteins, in addition to a type II thioesterase (TEII). Immediately downstream is a set of genes for desosamine biosynthesis (des) and macrolide ring hydroxylation. The study suggests that Pik TEII plays a role in forming a metabolic branch through which polyketides of different chain length are generated, and the glycosyl transferase (encoded by desVII) has the ability to catalyze glycosylation of both the 12- and 14-membered ring macrolactones. Moreover, the pikC-encoded P450 hydroxylase provides yet another layer of structural variability by introducing regiochemical diversity into the macrolide ring systems. The data support the notion that the architecture of the pik gene cluster as well as the unusual substrate specificity of particular enzymes contributes to its ability to generate four macrolide antibiotics.

Anti-Bacterial Agents↗
PubMed1998

The comparative amino acid sequences, substrate specificities and gene or cDNA nucleotide sequences of some prokaryote and eukaryote amidinotransferases: implications for evolution.

The amino acid sequences of the amidinotransferases and the nucleotide sequences of their genes or cDNA from four Streptomyces species (seven genes) and from the kidneys of rat, pig, human and human pancreas were compared. The overall amino acid and nucleotide sequences of the prokaryotes and eukaryotes were very similar and further, three regions were identified that were highly identical. Evidence is presented that there is virtually zero chance that the overall and high identity regions of the amino acid sequence similarities and the overall nucleotide sequence similarities between Streptomyces and mammals represent random match. Both rat and lamprey amidinotransferases were able to use inosamine phosphate, the amidine group acceptor of Streptomyces. We have concluded that the structure and function of the amidinotransferases and their genes has been highly conserved through evolution from prokaryotes to eukaryotes. The evolution has occurred with: (1) a high degree of retention of nucleotide and amino acid sequences; (2) a high degree of retention of the primitive Streptomyces guanine + cytosine (G + C) third codon position composition in certain high identity regions of the eukaryote cDNA; (3) a decrease in the specificities for the amidine group acceptors; and (4) most of the mutations silent in the regions suggested to code for active sites in the enzymes.

Amidinotransferases↗
PubMed1998

Hydroxylation of macrolactones YC-17 and narbomycin is mediated by the pikC-encoded cytochrome P450 in Streptomyces venezuelae.

BACKGROUND: . Streptomyces venezuelae produces two groups of antibiotics that include the 12-membered ring macrolides methymycin and neomethymycin, and the 14-membered ring macrolide pikromycin. Methymycin and pikromycin are derived from the corresponding precursors, YC-17 and narbomycin, respectively, by hydroxylation of the tertiary carbon position (C-10 in YC-17 or C-12 in narbomycin) on the macrolactone ring. In contrast, neomethymycin is derived from YC-17 by hydroxylation of the secondary carbon (C-12) of the propionyl starter unit sidechain. RESULTS: . Using a genetic and biochemical approach we have characterized a single P450 hydroxylase (PikC) in the methymycin/pikromycin biosynthetic gene cluster (pik) from S. venezuelae. Inactivation of pikC abolished production of all hydroxylated macrolides, with corresponding accumulation of YC-17 and narbomycin in the culture medium. The enzyme was produced efficiently and purified as a His-tagged protein from recombinant Escherichia coli cells. Purified PikC effectively converts YC-17 into methymycin and neomethymycin and narbomycin into pikromycin in vitro. CONCLUSIONS: . These results demonstrate that PikC is responsible for the conversion of YC-17 to methymycin and neomethymycin, and narbomycin to pikromycin in S. venezuelae. This substrate flexibility is unique and represents the first example of a P450 hydroxylase that can accept 12- and 14-membered ring macrolides as substrates, as well as functionalize at two positions on the macrolactone system. The broad substrate specificity of PikC provides a potentially valuable entry into the construction of novel macrolide- and ketolide-based antibiotics.

Amino Acid Sequence↗
PubMed1997

Characterization of a mitomycin-binding drug resistance mechanism from the producing organism, Streptomyces lavendulae.

In an effort to characterize the diversity of mechanisms involved in cellular self-protection against the antitumor antibiotic mitomycin C (MC), DNA fragments from the producing organism (Streptomyces lavendulae) were introduced into Streptomyces lividans and transformants were selected for resistance to the drug. Subcloning of a 4.0-kb BclI fragment revealed the presence of an MC resistance determinant, mrd. Nucleotide sequence analysis identified an open reading frame consisting of 130 amino acids with a predicted molecular weight of 14,364. Transcriptional analysis revealed that mrd is expressed constitutively, with increased transcription in the presence of MC. Expression of mrd in Escherichia coli resulted in the synthesis of a soluble protein with an Mr of 14,400 that conferred high-level cellular resistance to MC and a series of structurally related natural products. Purified MRD was shown to function as a drug-binding protein that provides protection against cross-linking of DNA by preventing reductive activation of MC.

Amino Acid Sequence↗
PubMed1996

Inducible synthesis of the mitomycin C resistance gene product (MCRA) from Streptomyces lavendulae.

The mcr locus from Streptomyces lavendulae confers high level resistance (> 100 micrograms/ml) to mitomycin C (MC) and related mitomycins when cloned into Streptomyces lividans. Production of the mcrA gene product (MCRA) was shown to be MC-inducible by identification of MCRA (M(r) of 54 kDa) using Western blot analysis and enzyme linked immunosorbent assay (ELISA). The magnitude of MCRA production was dependent on MC concentration, with primary induction starting at 0.1 microgram/ml and maximum induction at 10 micrograms/ml of the drug. Different levels of MCRA production were observed when other mitomycin metabolites were used as inducers, and the level of induction related directly to aziridine ring substitution on the individual molecules. Moreover, inducible synthesis of the mcr A gene product was unique to this structural class since production of MCRA did not occur as a general response to DNA damaging agents. The time profile of intracellular MCRA synthesis correlated with MC production in S. lavendulae, suggesting coordinated regulation of MC resistance and biosynthetic genes.

Antibiotics, Antineoplastic↗
PubMed1996

Metabolic engineering of cephalosporin biosynthesis in Streptomyces clavuligerus.

The biosynthesis of beta-lactams is one of the most thoroughly studied antibiotic pathways. The availability of the characteristics and the time profiles of activities of enzymes involved in the biosynthesis allows one to critically evaluate the potential rate-limiting steps in its production. Our approach to understanding the control of beta-lactam biosynthesis has been pursued using a two-stage strategy: (1) to predict the rate-limiting steps using a kinetic model and (2) to relax the rate-limiting steps by engineering the biosynthetic pathway or by altering the kinetic parameters of the predicted key rate-limiting enzyme. Kinetic analysis of the pathway dynamics of cephamycin C production in Streptomyces clavuligerus was performed using data obtained from wild type. Sensitivity analysis revealed that the availability of precursor alpha-aminoadipic acid and activity of ACV synthetase were the potential rate-limiting steps. Relaxation of the precursor limitation was accomplished by integration of an additional copy of the gene encoding lysine-epsilon-aminotransferase (lat) into the chromosome. The recombinant strain showed an increased level of cephamycin C production as expected. The intracellular levels of different intermediates in the pathway in batch cultures were analyzed.

Amino Acid Sequence↗
PubMed1996

Expression and analysis of a bacterial poly(hydroxyalkanoate) synthase in insect cells using a baculovirus system.

An improved method for expression of poly-beta-hydroxyalkanoate (PHA) synthase from Alcaligenes eutrophus has been developed using the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) in BTI-TN-5B1-4 Trichoplusia ni cells which results in high level production of active PHA synthase. Confirmation of expression of authentic PHA synthase was obtained by Western analysis which also revealed the presence of several apparent proteolytic cleavage products. N-terminal sequence data were obtained from the 64-kDa protein which verified its identity. The PHA synthase produced in this system constitutes approximately 50% of total protein after 60 h of viral infection and is found approximately equally distributed in both soluble and membrane-associated fractions. The expression level allowed rapid purification of the soluble form of PHA synthase to approximately 90% homogeneity in a single liquid chromatography step on hydroxylapatite. Using a direct spectrophotometric assay, analyses show that the enzyme has a pH optimum of 8.5, exhibits a concave-up Lineweaver-Burk plot, and a correlation between enzyme concentration and specific activity. Over 1000 units of soluble enzyme were obtained from a 250-ml culture of T. ni cells with an apparent initial specific activity of 12 mumol min-1 mg-1. The amount of PHA synthase activity is significantly higher than previously obtained from much larger bacterial cultures. The method described here should provide a general approach for the expression of active PHA synthases from a variety of bacterial sources to facilitate substrate specificity and mechanistic studies of these intriguing proteins.

Acyl Coenzyme A↗
PubMed1996

Production of a polyhydroxyalkanoate biopolymer in insect cells with a modified eucaryotic fatty acid synthase.

A novel pathway for the synthesis of poly-3-hydroxybutyrate has been engineered by simultaneous delivery of two genes into insect cells (Spodoptera frugiperda) by use of individual baculovirus vectors. This system includes expression of a dehydrase-domain mutant rat fatty acid synthase cDNA and the phbC gene encoding polyhydroxyalkanoate synthase from Alcaligenes eutrophus. The dehydrase-deficient fatty acid synthase provides de novo synthesis of R-(-)-3-hydroxybutyryl-coenzyme A as a premature termination product rather than palmityl-coenzyme A, the normal product of wild-type rat fatty acid synthase. High levels of this mutant multifunctional protein provide a suitable precursor pool of R-(-)-3-hydroxybutyryl-coenzyme A for conversion to poly-3-hydroxybutyrate in insect cells coexpressing the phbC gene product. This strategy for redesigning a poly-3-hydroxybutyrate biosynthetic pathway suggests a new method for generating structurally diverse polyhydroxyalkanoates by metabolic engineering.

Animals↗
PubMed1995

Polyketide synthase acyl carrier proteins from Streptomyces: expression in Escherichia coli, purification and partial characterisation.

Acyl carrier proteins (ACPs) of the type II polyketide synthases for the aromatic antibiotics actinorhodin, granaticin, frenolicin and oxytetracycline were expressed in Escherichia coli downstream of an inducible phage T7 promoter. For the act and otc genes, several of the first eight codons were changed to synonymous codons used in highly expressed E. coli genes. Correlated with these changes, the amounts of the act and otc ACPs purified from the recombinant E. coli cultures were an order of magnitude greater than for the gra and fren ACPs expressed from the unmodified genes. Electrospray mass spectrometry (ESMS) of the purified proteins confirmed their calculated M(r) based on the DNA sequences while also revealing that, in the act and gra ACP samples, some 2% and 30% of the holo-form of the protein was present (i.e., carrying the 4'-phosphopantetheine prosthetic group), with the remainder (and 100% of the otc and fren samples) being in the apo-form. Increasing incubation time post heat induction led to an increase in act holo-ACP. The recombinant act and gra ACPs could function in vitro as substrates for an S. coelicolor malonyl CoA:ACP acyl transferase, as measured by the coupling of a labelled malonyl unit to the ACP; their quantitative abilities to do so correlated with the proportions of deduced holo form in the two samples.

Acyl Carrier Protein↗
PubMed1995

Effects of enhanced lysine epsilon-aminotransferase activity on cephamycin biosynthesis in Streptomyces clavuligerus.

A recombinant strain of S. clavuligerus (LHM100) that contains an additional copy of the gene (lat) encoding lysine epsilon-aminotransferase (LAT) was analyzed and compared to the wild-type for intracellular concentrations of primary metabolites involved in cephamycin C biosynthesis. This strain had been shown previously to produce higher levels of the antibiotic because of increased levels of LAT, a rate-limiting enzyme involved in the production of alpha-aminoadipic acid. The results showed that the overall growth kinetics of the two strains were comparable, including the intracellular concentrations of cysteine, valine and lysine. In contrast, 60% higher antibiotic production was observed in LHM100, which reflected a significant temporal variation in specific metabolite production rate. The time profile of LAT activity was consistently higher in LHM100; however, alpha-aminoadipic acid levels showed unexpected variation during the growth cycle. These results support the proposal that rate-limiting enzymes in cephamycin C biosynthesis are temporally controlled, and indicate that optimization of metabolite production will require differential overexpression of several biosynthetic genes.

Cephamycins↗
PubMed1995

Heterologous expression of an engineered biosynthetic pathway: functional dissection of type II polyketide synthase components in Streptomyces species.

Polyketides are an extensive class of secondary metabolites with diverse molecular structures and biological activities. A plasmid-based multicomponent polyketide synthase expression cassette was constructed using a subset of actinorhodin (act) biosynthetic genes (actI-orf1, actI-orf2, actI-orf3, actIII, actVII, and actIV) from Streptomyces coelicolor which specify the construction of the anthraquinone product aloesaponarin II, a molecule derived from acetyl coenzyme A and 7 malonyl coenzyme A extender units. This system was designed as an indicator pathway in Streptomyces parvulus to quantify polyketide product formation and to examine the functional significance of specific polyketide synthase components, including the act beta-ketoacyl synthase (beta-KS; encoded by actI-orf1 and actI-orf2) and the act cyclase/dehydrase (encoded by actVII-orf4). Site-directed mutagenesis of the putative active site Cys (to a Gln) in the actI-orf1 beta-KS product completely abrogated aloesaponarin II production. Changing the putative acyltransferase active-site Ser (to a Leu) located in the actI-orf1 beta-KS product led to significantly reduced but continued production of aloesaponarin II. Replacement of the expression cassette with one containing a mutant form of actI-orf2 gave no production of aloesaponarin II or any other detectable polyketide products. However, an expression cassette containing a mutant form of actVII-orf4 gave primarily mutactin with low-level production of aloesaponarin II.

Anthraquinones↗
PubMed1994

Cloning, sequencing and deduced functions of a cluster of Streptomyces genes probably encoding biosynthesis of the polyketide antibiotic frenolicin.

A 10.2-kb fragment of DNA from Streptomyces roseofulvus, which contains polyketide synthase (PKS)-encoding genes (fren) presumed to determine production of the antibiotics frenolicin and the nanaomycins, was cloned. A 5530-bp continuous segment of this DNA was sequenced. Analysis of the sequence revealed five complete open reading frames (ORFs) transcribed in one direction (ORFs 1, 2, 3, 5, 4) and one (ORFX), located between ORF3 and ORF5, transcribed in the opposite direction. The deduced amino-acid sequences of ORFs 1, 2, 3, 4 and 5 closely resemble the sequences of known components of the type-II PKS from other Streptomyces species: putative heterodimeric (ORF1 + 2) ketosynthase, acyl carrier protein, cyclase and ketoreductase, respectively. A resemblance between the N-terminal and C-terminal halves of the ORF4 product--also discovered in the corresponding genes from other isochromanequinone antibiotic producers--suggests a possible origin of the cyclase-encoding gene by duplication. ORFX appears to represent a novel class of genes of unknown function present not only in the fren cluster, but also in other clusters of aromatic antibiotic biosynthetic genes in Streptomyces species. The fren-ORF1-5 genes, encoding a PKS that constructs a nascent polyketide of either 16 or 18 carbons, compared with fixed lengths of 16 and 20 for other available examples, are proving to be valuable for understanding the mechanisms controlling polyketide chain length and patterns of reduction and cyclisation.

Acyltransferases↗
PubMed1994

Sequences of the oxytetracycline polyketide synthase-encoding otc genes from Streptomyces rimosus.

The complete nucleotide sequences of the Streptomyces rimosus oxytetracycline (oxyTc) polyketide synthase (PKS)-encoding genes (otcY) has been determined, revealing three open reading frames. The deduced amino-acid sequences correspond to the presumed heterodimeric beta-ketoacyl synthase and acyl carrier protein found in other type-II (multicomponent) PKS systems that specify construction of acetate-derived polyketide antibiotics.

Amino Acid Sequence↗
PubMed1994

Analysis of type II polyketide beta-ketoacyl synthase specificity in Streptomyces coelicolor A3(2) by trans complementation of actinorhodin synthase mutants.

Complementation of defined actinorhodin beta-ketoacyl synthase (KS) mutants by various other KS genes suggested that the ORF1-encoded KS may be relatively generalized in function, whereas the ORF2-encoded KS component may provide specificity in polyketide chain construction. Evidence for differential temporal-spatial expression of the actinorhodin and spore pigment KSs in Streptomyces coelicolor was obtained.

Animals↗
PubMed1993

Precursor flux control through targeted chromosomal insertion of the lysine epsilon-aminotransferase (lat) gene in cephamycin C biosynthesis.

Targeted gene insertion methodology was used to study the effect of perturbing alpha-aminoadipic acid precursor flux on the overall production rate of beta-lactam biosynthesis in Streptomyces clavuligerus. A high-copy-number plasmid containing the lysine epsilon-aminotransferase gene (lat) was constructed and used to transform S. clavuligerus. The resulting recombinant strain (LHM100) contained an additional complete copy of lat located adjacent to the corresponding wild-type gene in the chromosome. Biological activity and production levels of beta-lactam antibiotics were two to five times greater than in wild-type S. clavuligerus. Although levels of lysine epsilon-aminotransferase were elevated fourfold in LHM100, the level of ACV synthetase, whose gene is located just downstream of lat, remained unchanged. These data strongly support the notion that direct perturbation of alpha-aminoadipic acid precursor flux resulted in increased antibiotic production. This strategy represents a successful application of metabolic engineering based on theoretical predictions of precursor flux in a secondary metabolic pathway.

Base Sequence↗