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R McDaniel

Publications and source records attributed to R McDaniel.

34 records · Page 2Linked to original sources
PubMed1997

Engineered intermodular and intramodular polyketide synthase fusions.

BACKGROUND: Modular polyketide synthases (PKSs) are very large multifunctional enzyme complexes that synthesize a number of medicinally important natural products. The modular arrangement of active sites has made these enzyme systems amenable to combinatorial manipulation for the biosynthesis of novel polyketides. Here, we investigate the involvement of subunit interactions in hybrid and artificially linked PKSs with several series of intermodular and intramodular fusions using the erythromycin (6-deoxyerythronolide B synthase; DEBS) and rapamycin (RAPS) PKSs. RESULTS: Several two-module and three-module derivatives of DEBS were constructed by fusing module 6 to either module 2 or module 3 at varying junctions. Polyketide production by these intramodular fusions indicated that the core set of active sites remained functional in these hybrid modules, although the ketoreductase domain of module 6 was unable to recognize unnatural triketide and tetraketide substrates. Artificial trimodular PKS subunits were also engineered by covalently linking modules 2 and 3 of DEBS, thereby demonstrating the feasibility of constructing single-chain PKSs. Finally, a series of fusions containing DEBS and RAPS domains in module 2 of an engineered trimodular PKS revealed the structural and functional tolerance for hybrid modules created from distinct PKS gene clusters. CONCLUSIONS: The general success of the intermodular and intramodular fusions described here demonstrates significant structural tolerance among PKS modules and subunits and suggests that substrate specificity, rather than protein-protein interactions, is the primary determinant of molecular recognition features of PKSs. Furthermore, the ability to artificially link modules may considerably simplify the heterologous expression of modular PKSs in higher eukaryotic systems.

Amino Acid Sequence↗
PubMed1995

Rational design of aromatic polyketide natural products by recombinant assembly of enzymatic subunits.

Recent advances in understanding of bacterial aromatic polyketide biosynthesis allow the development of a set of design rules for the rational manipulation of chain synthesis, reduction of keto groups and early cyclization steps by genetic engineering. The concept of rational design is illustrated by the preparation of Streptomyces strains that produce two new polyketides by expression of combinations of appropriate enzymatic subunits from naturally occurring polyketide synthases. The potential for generating molecular diversity within this class of molecules by genetic engineering is enormous.

Genetic Engineering↗
PubMed1994

Engineered biosynthesis of novel polyketides: influence of a downstream enzyme on the catalytic specificity of a minimal aromatic polyketide synthase.

To identify the minimum set of polyketide synthase (PKS) components required for in vivo biosynthesis of aromatic polyketides, combinations of genes encoding subunits of three different aromatic PKSs--act from Streptomyces coelicolor A3(2) (an actinorhodin producer), fren from Streptomyces roseofulvus (a frenolicin and nanaomycin producer), and tcm from Streptomyces glaucescens (a tetracenomycin producer)--were expressed in a recently developed Streptomyces host-vector system. The "minimal" components (ketosynthase/putative acyltransferase, chain length-determining factor, and acyl carrier protein) were produced with and without a functional polyketide ketoreductase and/or cyclase, and the polyketide products of these recombinant strains were structurally characterized. Several previously identified polyketides were isolated in addition to two previously unidentified polyketides, dehydromutactin and SEK 15b, described here. The results proved that the act cyclase is not required for the biosynthesis of several aberrantly cyclized products that have been previously reported. They are also consistent with earlier conclusions that the minimal PKS controls chain length as well as the regiospecificity of the first cyclization and that it can do so in the absence of both a ketoreductase and a cyclase. However, the ability of the minimal tcm PKS to synthesize two different singly cyclized intermediates suggests that it is unable to accurately control the course of this reaction by itself. In the presence of a downstream enzyme, the flux through one branch of the cyclization pathway increases relative to the other. We propose that these alternative specificities may be due to the ability of downstream enzymes to associate with the minimal PKS and to selectively inhibit a particular branch of the cyclization pathway.

DNA Mutational Analysis↗
PubMed1994

Engineered biosynthesis of novel polyketides: stereochemical course of two reactions catalyzed by a polyketide synthase.

A genetically engineered strain expressing the essential components of the tetracenomycin polyketide synthase (tcm PKS) along with the actinorhodin ketoreductase (act KR) was found to produce two new (diastereomeric) aromatic polyketides, designated RM20b and RM20c, in addition to RM20, whose structure was reported earlier [McDaniel, R., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1993) Science 262, 1546-1550]. Spectroscopic and in vivo isotopic labeling analysis of RM20b and RM20c revealed that their polyketide backbones were identical to that of RM20 with respect to chain length, regiospecificity of ketoreduction, and regiospecificity of the first intramolecular aldol condensation. This is consistent with earlier predictions that the essential components of the PKS--a bifunctional ketosynthase/acyltransferase, a chain length determining factor, and an acyl carrier protein--are responsible for controlling each of these features of the polyketide backbone [McDaniel, R., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1993) Science 262, 1546-1550; McDaniel, R., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1993) J. Am. Chem. Soc. 115, 11671-11675; Fu, H., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1994) J. Am. Chem. Soc. 116, 4166-4170]. In addition, however, RM20b and RM20c possess two unusual features. In both molecules the hydroxyls on sp3 C-9 and C-7 of the first six-membered ring, which arise as a result of ketoreduction and aldol condensation, respectively, are intact, rather than being lost via dehydration. Furthermore, the relative yield of RM20b (in which these hydroxyls are syn) is 7-fold greater than that of RM20c (in which they are anti).(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Isotopes↗
PubMed1993

Engineered biosynthesis of novel polyketides.

Polyketide synthases (PKSs) are multifunctional enzymes that catalyze the biosynthesis of a huge variety of carbon chains differing in their length and patterns of functionality and cyclization. Many polyketides are valuable therapeutic agents. A Streptomyces host-vector system has been developed for efficient construction and expression of recombinant PKSs. Using this expression system, several novel compounds have been synthesized in vivo in significant quantities. Characterization of these metabolites has provided new insights into key features of actinomycete aromatic PKS specificity. Thus, carbon chain length is dictated, at least in part, by a protein that appears to be distinctive to this family of PKSs, whereas the acyl carrier proteins of different PKSs can be interchanged without affecting product structure. A given ketoreductase can recognize and reduce polyketide chains of different length; this ketoreduction always occurs at the C-9 position. The regiospecificity of the first cyclization of the nascent polyketide chain is either determined by the ketoreductase, or the chain-extending enzymes themselves. However, the regiospecificity of the second cyclization is determined by a distinct cyclase, which can discriminate between substrates of different chain lengths.

Base Sequence↗
PubMed1993

Conditions for glutamate dehydrogenase activity in heart mitochondria.

Although heart mitochondria contain glutamate dehydrogenase, it has not been thought to play a role in their metabolism. We investigated this matter to define the conditions under which it is active. We found modest activity in the presence of glutamate and malate and a continuous source of ADP when pyruvate is added. This increases several fold as the osmolarity is increased from 296 to 370 mosM. At the higher osmolarity ammonia formation is brief, associated with a lower intramitochondrial alpha-ketoglutarate from citrate does not make up for the drop in glutamate conversion to alpha-ketoglutarate. Mitochondrial content of nucleotides and CoA compounds are not altered by pyruvate addition. The rate of glutamate deamination by GDH in sonicated heart mitochondria agrees with the rate of ammonia formation in intact mitochondria in the presence of pyruvate (20 nmol/min/mg of mitochondrial protein). We conclude pyruvate lowers mitochondrial oxalacetate which decreases alpha-ketoglutarate formation by transamination. The lower mitochondria alpha-ketoglutarate level permits glutamate deamination until alpha-ketoglutarate reaches a level that inhibits the forward reaction. Further proof of the key role of alpha-ketoglutarate is seen with aminooxyacetate which blocks transamination. In its presence ammonia formation occurs at the same rate (18 nm/min/mg of mitochondrial protein), is not dependent upon pyruvate, and does not stop after a couple of minutes. Leucine, which decreases alpha-ketoglutarate inhibition of GDH, also results in ammonia formation, further supporting the concept of regulation by alpha-ketoglutarate. The higher osmolarity increases GDH activity by increasing alpha-ketoglutarate transport from mitochondria.

Adenosine Diphosphate↗
PubMed1993

Genetic construction and functional analysis of hybrid polyketide synthases containing heterologous acyl carrier proteins.

The gene that encodes the acyl carrier protein (ACP) of the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2) was replaced with homologs from the granaticin, oxytetracycline, tetracenomycin, and putative frenolicin polyketide synthase gene clusters. All of the replacements led to expression of functional synthases, and the recombinants synthesized aromatic polyketides similar in chromatographic properties to actinorhodin or to shunt products produced by mutants defective in the actinorhodin pathway. Some regions within the ACP were also shown to be interchangeable and allow production of a functional hybrid ACP. Structural analysis of the most abundant polyketide product of one of the recombinants by electrospray mass spectrometry suggested that it is identical to mutactin, a previously characterized shunt product of an actVII mutant (deficient in cyclase and dehydrase activities). Quantitative differences in the product profiles of strains that express the various hybrid synthases were observed. These can be explained, at least in part, by differences in ribosome-binding sites upstream of each ACP gene, implying either that the ACP concentration in some strains is rate limiting to overall PKS activity or that the level of ACP expression also influences the expression of another enzyme(s) encoded by a downstream gene(s) in the same operon as the actinorhodin ACP gene. These results reaffirm the idea that construction of hybrid polyketide synthases will be a useful approach for dissecting the molecular basis of the specificity of PKS-catalyzed reactions. However, they also point to the need for reducing the chemical complexity of the approach by minimizing the diversity of polyketide products synthesized in strains that produce recombinant polyketide synthases.

Acyl Carrier Protein↗
PubMed1993

Antigen nonspecific effect of major histocompatibility complex haplotype on autoantibody levels in systemic lupus erythematosus-prone lpr mice.

MHC-linked genes strongly influence susceptibility to autoimmune diseases and also regulate responses to exogenous antigens. To begin to understand the mechanism of this MHC effect on disease, we have investigated MHC-congenic mouse strains that develop spontaneous autoimmunity because of the lpr gene. C57BL6/lpr (B6/lpr) mice (H-2b) are known to have substantial levels of autoantibodies to chromatin, single stranded DNA (ssDNA3), and IgG of different murine subclasses (rheumatoid factor). We have crossed the H-2d and the H-2bm12 (la mutant) haplotypes onto the B6/lpr background. Surprisingly, levels of all the autoantibodies were markedly lower in B6/lpr.H-2d, but levels in B6/lpr.H-2bm12 were no different from those in B6/lpr mice. The downregulating influence of the H-2d allele was dominant, and there was no effect on autoantibody fine specificities. The genetics of the H-2d effect and its diffuse influence on multiple autoantibody specificities, in addition to the lack of effect of the bm12 mutation, which modifies the peptide-binding groove of I-A, together raise the question of whether MHC-linked genes other than classical (IR) genes may be responsible for MHC disease associations in this model.

Animals↗
PubMed1985

The interaction of calcium with gangliosides in bilayer membranes.

We studied the binding of calcium to bilayer membranes formed from mixtures of phosphatidylcholine and mono-, di-, or trisialoganglioside by measuring its effect on the electrophoretic mobility of multilamellar vesicles and the conductance of planar bilayers. In 0.001 M monovalent salt solutions the surface potential of the membranes is large and micromolar concentrations of calcium have a significant effect on the mobility and conductance. In 0.1 M monovalent salt solutions the surface potential is small and millimolar concentrations of calcium are required to affect these parameters. The strong apparent binding of calcium we observed at low ionic strength could be due to the nonspecific accumulation of calcium in the electrical diffuse double layer. To distinguish between this nonspecific effect and binding of calcium to the membrane, we substituted dimethonium for calcium. Dimethonium is a divalent cation that screens negative charges but does not bind to lipids. We also examined the effect of replacing phosphatidylcholine by monoolein: calcium binds to phosphatidylcholine but not to monoolein. We describe our electrophoretic mobility results by combining the Poisson-Boltzmann and Navier-Stokes equations with the Langmuir adsorption isotherm. We conclude that calcium binds weakly to gangliosides with an intrinsic association constant of less than 100 M-1, which is similar to the association constant of calcium with phospholipids.

Binding Sites↗
PubMed1985

Interaction of gentamicin and spermine with bilayer membranes containing negatively charged phospholipids.

We measured the electrophoretic mobility of multilamellar phospholipid vesicles, the 31P NMR spectra of both sonicated and multilamellar vesicles, and the conductance of planar bilayer membranes to study the binding of spermine and gentamicin to membranes. Spermine and gentamicin do not bind significantly to the zwitterionic lipid phosphatidylcholine. We measured the concentrations of gentamicin and spermine that reverse the charge on vesicles formed from a mixture of phosphatidylcholine and either phosphatidylserine or phosphatidylinositol. From these measurements, we determined that the intrinsic association constants of the cations with these negative lipids are all about 10 M-1. This value is orders of magnitude lower than the apparent binding constants reported in the literature by other groups because the negative electrostatic surface potential of the membranes and the resultant accumulation of these cations in the aqueous diffuse double layer adjacent to the membranes have not been explicitly considered in previous studies. Our main conclusion is that the Gouy-Chapman-Stern theory of the aqueous diffuse double layer can describe surprisingly well the interaction of gentamicin and spermine with bilayer membranes formed in a 0.1 M NaCl solution if the negative phospholipids constitute less than 50% of the membrane. Thus, the theory should be useful for describing the interactions of these cations with the bilayer component of biological membranes, which typically contain less than 50% negative lipids. For example, our results support the suggestion of Sastrasinh et al. [Sastrasinh, M., Krauss, T. C., Weinberg, J. M., & Humes, H. D. (1982) J. Pharmacol. Exp. Ther. 222, 350-358] that phosphatidylinositol is the major binding site for gentamicin in renal brush border membranes.

Electric Conductivity↗
PubMedDate not supplied

Fatigue and quality of life outcomes of exercise during cancer treatment.

PURPOSE: Despite the recognition of fatigue as a common and distressing symptom during cancer treatment, there are few evidence-based interventions available to manage such fatigue. The purpose of this multi-institutional pilot study was to explore the effects of a home-based moderate walking exercise intervention on fatigue, physical functioning, emotional distress, and quality of life (QOL) during breast cancer treatment. DESCRIPTION OF STUDY: Fifty-two women were recruited from five university hospital outpatient departments for this pilot study with an experimental design. Subjects were randomly assigned to the walking program or to usual care during adjuvant chemotherapy or radiation therapy for breast cancer. Symptoms, physical functioning, and QOL were measured at baseline, midtreatment, and at the end of treatment. RESULTS: Women who exercised at least 90 minutes per week on 3 or more days reported significantly less fatigue and emotional distress as well as higher functional ability and QOL than women who were less active during treatment. CLINICAL IMPLICATIONS: A home-based walking exercise program is a potentially effective, low-cost, and safe intervention to manage fatigue and to improve QOL during adjuvant chemotherapy or radiation therapy for breast cancer. This health-promoting self-care activity needs further testing in large randomized clinical trials.

Activities of Daily Living↗
PubMedDate not supplied

Collection and preparation of sidestream cigarette smoke for trace elemental determinations by graphite furnace atomic absorption spectrometry and inductively coupled plasma mass spectrometry.

A novel method for the collection and preparation of sidestream cigarette smoke condensate is described for trace elemental analysis by inductively coupled plasma mass spectrometry and graphite furnace atomic absorption spectrometry. The smoke collection method utilizes a specially designed chimney that collects and directs the sidestream smoke (SS) to a 2-stage trapping system consisting of an impaction trap followed by a 0.8 microm mixed cellulose ester filter. The samples are digested with nitric acid in a commercial heating block before analysis. The method limits of detection (LODs) are 1, 0.2, 2, 9, 6, and 7 ng/cigt for As, Cd, Pb, Ni, Se, and Cr, respectively. The SS collected from an industry reference cigarette, 1R4F, produced by the University of Kentucky was analyzed. The concentrations of As, Cd, and Pb in 1R4F were determined to be 27.3+/-2.1, 412+/-14, and 43.8+/-2.0 ng/cigt, respectively, while the concentrations of Ni, Cr, and Se are below the method LOD. Consequently, this novel method successfully addresses contamination, instrumentation, and collection issues for performing trace elemental analysis of sidestream cigarette smoke condensate.

Mass Spectrometry↗