Browsing by Author "Parry, Ronald J."
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Item Biosynthetic studies of coronatine and polyoximic acid(1993) Walker, Alan Edward; Parry, Ronald J.Coronatine (1) (Fig 1) is a novel, non-host specific phytotoxin elaborated in the liquid fermentation broths of several different Pseudomonas syringae pathovers. Infection of tomato plants by P. syringae pv. $tomato,\sp1$ Italian ryegrass by P. syringae pv. $atropurpurea,\sp{2,3}$ soybean by P. syringae pv. $glycinae,\sp{3,4}$ and Prunus spp. by P. syringae pv. $morsprunorum\sp3$ induces yellow chlorotic haloes, stunting, and plant tissue hypertrophy due to the production of coronatine.$\sp{2,4}$ Coronatine presents two distinct biosynthetic problems, one being the origin of coronafacic acid (2) the other the origin of the novel amino acid coronamic acid (3) (Fig 1). The investigations presented in this dissertation were directed towards an understanding of the biosynthetic pathway to coronamic acid. Our results from the administration of specifically labeled precursors to Pseudomonas syringae pv. glycinae (PDCC # 4182) suggest that: (1) the retention of the tritium label from (3-$\sp3$H) -L-alloisoleucine rules out the intermediacy of 3-methylene-norvaline on the biosynthetic pathway to coronamic acid, (2) the incorporation of approximately half of the $\sp{15}$N label from a mixture of (2-$\sp{13}$C, $\sp{15}$N)-DL-isoleucine and (2-$\sp{13}$C, $\sp{15}$N)-DL-alloisoleucine indicates that the cyclisation reaction leading to the formation of coronamic acid from L-alloisoleucine does not involve the loss of nitrogen and (3) the incorporation of (4-$\sp2$H$\sb1$,5-$\sp2$H$\sb1$)-($\pm$)-coronamic acid provides some experimental evidence in support of the hypothesis that coronamic acid and coronafacic acid are biosynthesised separately and coupled by the formation of an amide bond to give coronatine. Polyoximic acid (8) (Fig 4) is the amino acid component of polyoxins A, F, H, I, and K, a unique class of antifungal antibiotics elaborated in the fermentation broths of Streptomyces cacaoi var. $asoensis.\sp{20,22,37}$ Our investigations presented in this dissertation on the biosynthetic origin of polyoximic acid by the administration of specifically labeled precursors to Streptomyces cacaoi var. asoensis (ATCC # 19094) suggest that: (1) polyoximic acid is biosynthetically derived from L-isoleucine, (2) the incorporation of the $\sp{13}$C label from (1-$\sp{13}$C)-L-alloisoleucine into C-1" of polyoximic acid requires that the formation of the exocyclic double bond of polyoximic acid be formed by a syn-elimination of hydrogen from C-3 and not an anti-elimination as previously reported$\sp{38}$ and (3) the retention of two deuterium atoms, at C-4" of polyoximic acid derived from (6-$\sp{13}$C,$\sp2$H$\sb3$)-DL-isoleucine and (6-$\sp{13}$C,$\sp2$H$\sb3$)-DL-alloisoleucine, indicates that the cyclisation reaction requires the loss of only one hydrogen atom from L-isoleucine and L-alloisoleucine.Item Chemical and biological approaches to understanding the biosynthesis of the Pseudomonas polyketide coronafacic acid(2000) Jiralerspong, Sao; Parry, Ronald J.Coronatine is a phytotoxin produced by Pseudomonas syringae spp. that infect a number of commercially important crops. Coronatine is composed of a cyclopropyl amino acid, coronamic acid, and a novel polyketide, coronafacic acid (CFA). This work discloses (1) chemical and (2) biological approaches towards understanding CFA biosynthesis. 1. It was previously known that CFA is assembled from a pyruvate, a butyrate, and three acetate units, but the mode of pyruvate incorporation was obscure. Precursor incorporation experiments with labeled forms of glutamate have now shown that pyruvate is incorporated only after its conversion to one of the starter units, alpha-ketoglutarate, succinic semialdehyde, or glutamate. It was hypothesized that polyketide chain extension of one of these starter units with an acetate unit followed by first ring cyclization would give 2-cyclopenten-1-one 2-carboxylic acid (CPC). This could be extended with a butyrate unit to give 2-[1-oxo-2-cyclopenten-2-ylmethyl]butanoic acid (CPE), a compound previously isolated from P. syringae culture broths. Finally, chain extension with an acetate unit and closure of the second ring would give CFA. This model of CFA biosynthesis (starter unit → CPC → CPE → CFA) has been tested by precursor incorporation experiments involving labeled forms of succinic semialdehyde and CPC. In addition, several synthetic routes to CPE have also been explored. 2. The nucleotide sequence of the remaining unsequenced region of the CFA biosynthetic gene cluster was completed in this work. Analysis revealed the presence of two large ORFs, cfa6 and cfa7, whose gene products show homologies to type I polyketide synthases (PKSs). Previous work had demonstrated the presence of a type II PKS in the CFA cluster, including two adenylating enzymes (Cfl and Cfa5) and a thioesterase (Cfa9). Consideration of all the putative enzymatic activities of the CFA cluster (Cfl and Cfa1--Cfa9) has allowed a refinement of the earlier biosynthetic model, in which a good correlation exists between the enzymatic activities present and the putative biochemical steps of the earlier model (starter unit → CPC → CPE → CFA). Further characterization of the CFA cluster has begun with the soluble expression and partial purification of the Cfl and Cfa5 proteins as maltose binding protein fusions.Item Investigation of valanimycin biosynthesis(1996) Li, Wenying; Parry, Ronald J.The antibiotic valanimycin is an azoxy compound produced by Streptomyces viridifaciens MG456-hF10. Precursor incorporation experiments with (4-$\sp{13}$C) -N-(isobutylamino)serine and $\rm \lbrack\sp{15}N\sb2\rbrack$-N-(isobutylamino)serine suggest that N-(isobutylamino)serine is a specific precursor of valanimycin. Searching for enzymes related to the biosynthetic pathway of valanimycin led to the identification of valine decarboxylase and isobutylamine N-hydroxylase in crude extracts of S. viridifaciens MG456-hF10 cells. Isobutylamine N-hydroxylase requires FAD and NADH for activity. The enzyme was found to consist of two protein components. The A component of isobutylamine N-hydroxylase was partially purified and the B component was purified to homogeneity. The properties of isobutylamine N-hydroxylase were studied. Enzyme replacement experiments suggested that the A component catalyzes the reduction reaction of FAD or FMN with NADH to form FADH$\sb2$ or FMNH$\sb2,$ and that the B component catalyzes the hydroxylation reaction of isobutylamine with the aid of FADH$\sb2$ or FMNH$\sb2,$ and molecular oxygen. Attempts to clone the B component of isobutylamine N-hydroxylase were made. A library of S. viridifaciens MG456-hF10 genomic DNA was constructed using a Lambda DASH II phage vector. The library was screened with a oligonucleotide probe derived from the N-terminal protein sequence of the B component of isobutylamine N-hydroxylase. No gene fragment corresponding to the N-terminal sequence was found. Further screening will be necessary.Item Investigations of neplanocin a reductase, an enzyme involved in aristeromycin biosynthesis(1998) Jiang, Yijia; Parry, Ronald J.The nucleoside antibiotics aristeromycin and neplanocin A are two naturally occurring carbocyclic analogs of adenosine produced by Streptomyces citricolor. Partial purification of the crude cell-free extracts of S. citricolor by DEAE chromatography, ammonium sulfate fractionation, and chromatography on phenyl agarose yielded a cell-free system that converted neplanocin A to aristeromycin as judged by HPLC and NMR analysis. The production of aristeromycin was dependent upon the addition of NADPH to the incubation mixture and $\rm NAD\sp+$ was found to increase the production of aristeromycin at very low concentration when combined with NADPH. The apparent $\rm K\sb{m}\sp{s}$ of neplanocin A and NADPH were determined to be 15.4 $\rm\mu M$ and 11.1 $\rm\mu M$ for neplanocin A reductase using phenyl agarose purified enzyme. The native molecular weight of the reductase is around 50 KDa when measured by gel filtration chromatography and the pl of the enzyme is 4.8. The mechanism of the reduction reaction was explored by incubation of neplanocin A with isotopically labeled NADPH and partially purified reductase, and by incubation of the enzyme with NADPH in a deuterated buffer. The mechanism of the double bond reduction was further studied with the substrate analogs $4\sp\prime$-deshydroxymethyl- and $3\sp\prime$-deoxy-neplanocin A, and by isotope exchange experiments with aristeromycin and neplanocin A in deuterated buffer containing $\rm NAD\sp+.$ The results of these investigations will be presented in this thesis.Item Investigations of the biosynthesis of sinefungin(1989) Arzu, Isidora Yvonne; Parry, Ronald J.The biosynthesis of the antifungal, antiviral antiobiotic, sinefungin (1), produced by Streptomyces griseolus has been investigated. Precursor incorporation studies using (U-$\sp{14}$C), (5-$\sp3$H), (5-$\sp{13}$C) and (5-$\sp{13}$C, 5-$\sp{15}$N)-ornithine have shown that carbons 6$\sp\prime$-10$\sp\prime$ were derived from the intact incorporation of ornithine with retention of the C-5 nitrogen and with the loss of one of the protons from C-5 of ornithine. The results of the feeding experiments of ($\sp{14}$C) and ($\sp3$H) labelled adenosine have indicated that the adenine moiety of adenosine was incorporated intact. Administration of adenosine labelled on the ribose moiety indicated that adenosine was incorporated into sinefungin with 50% loss of protons from C-5$\sp\prime$ of adenosine.Item Investigations of the biosynthesis of sparsomycin(1988) Gomez, Mary Elizabeth Eudy; Parry, Ronald J.Investigations of the biosynthesis of the antitumor antibiotic sparsomycin (1) by Streptomyces sparsogenes have been carried out. Incorporation studies employing ($\sp{13}$C)-labeled precursors have shown that the monoxodithioacetal moiety (6) of the antibiotic arises from the step-wise introduction of a thiomethyl group into the S-methyl group of S-methyl-D-cysteine. The methyl group of (6) has its origin in the methyl group of L-methionine, but an experiment utilizing (methyl-$\sp3$H,$\sp{35}$S) -L-methionine has demonstrated that intact incorporation of the thiomethyl group of this precursor does not occur. The results of feeding experiments with ($\sp{13}$C)- and ($\sp2$H)-labeled forms of tryptophan have indicated that the uracil moiety (7) of sparsomycin is derived from the indole nucleus of tryptophan via aromatic ring cleavage followed by recyclization. Preliminary evidence for the intermediacy of N-formyl-anthranilic acid in the conversion of tryptophan to sparsomycin has been obtained.Item Investigations of the biosynthesis of sparsomycin(1992) Li, Yan; Parry, Ronald J.The biosynthesis of the antitumor antibiotic, sparsomycin, produced by Streptomyces sparsogenes, has been investigated. Incorporation studies employing ($\sp{13}$C)-labeled precursors have shown that both L- and D-isomers of S-(methylthio)methyl-cysteine are specifically incorporated into the antibiotic. Furthermore, both L- and D-isomers of S-(methylthio)methylcysteinol are proved to be the advanced intermediates lying beyond S-(methylthio)methylcysteine. S-Methylcysteine is found not to be incorporated intact into the antibiotic; however, an isotopic trapping experiment and preliminary cell-free studies indicate that S-methylcysteine is still quite likely to be an intermediate on the pathway. A very high isotope effect was observed during studies of the loss of hydrogen atoms from the methyl group of S-methylcysteine. The uracil moiety (6) of sparsomycin is found to be derived from the indole fragment of tryptophan. N-Formylanthranilic acid was originally proposed as an intermediate lying beyond tryptophan on the pathway to 6. However, precursor incorporation studies have shown that N-formylanthranilic acid is incorporated by deformylation to anthranilic acid, which is then converted back to tryptophan before incorporation into sparsomycin. Therefore, the originally proposed pathway has to be revised. The final steps in the biosynthesis of uracil moiety are shown to be similar to the biosynthesis of xanthosine monophosphate (XMP).Item Mechanistic investigations of novel, irreversible inhibitors of S-adenosylhomocysteine hydrolase(1992) Muscate, Angelika; Parry, Ronald J.Isolation of bovine S-adenosylhomocysteine hydrolase by affinity chromatography unexpectedly yielded two forms of the enzyme. Type A contained 4 moles of NAD$\sp+$/mole of enzyme tetramer, while Type B had only half that number of nucleotide cofactors associated with it. The acetylenic analog of adenosine, 9-(5$\sp\prime$,6$\sp\prime$-dideoxy-$\beta$-D-ribo-hex-5$\sp\prime$-ynofuranosyl)-adenine, was synthesized, and its behavior as an inhibitor of Type A enzyme examined. Irreversible inactivation of the enzyme with excess inhibitor was accompanied by the reduction of 2 equivalents of NAD$\sp+$ to NADH, release of the remaining NAD$\sp+$ from the enzyme, and binding of 4 equivalents of inhibitor per enzyme tetramer. Denaturation studies suggested that two equivalents of the inhibitor may form a covalent bond between the enzyme and the oxidized inhibitor. The inactivation behavior exhibited by Type A and Type B enzyme with 2$\sp\prime$-deoxy-2$\sp\prime$,2$\sp\prime$-difluoroadenosine was compared. The Type B enzyme was irreversibly inactivated by excess inhibitor with concurrent reduction of 1 mole of NAD$\sp+$ to NADH and release of 1 mole of NAD$\sp+$. Inactivation of the Type A enzyme was accompanied by the reduction of 1 mole of NAD$\sp+$ to NADH and release of ca. 2 moles of NAD$\sp+$. Upon dialysis, 50% of the original activity of the enzyme was regained. Binding stoichiometries of 1 and 3 moles inhibitor/mole of enzyme tetramer were determined for the Type B and Type A enzyme, respectively. Denaturation studies indicated that 1 equivalent of the oxidized inhibitor may have formed a covalent bond with the Type A enzyme, but not with Type B. The binding behavior of adenosine with both types of the enzyme was also studied. Type B enzyme exhibited a large binding affinity of 4-25 moles of adenosine/mole of enzyme tetramer, while Type A bound only 3 moles of adenosine/mole enzyme tetramer. The binding of adenosine to the Type B enzyme was accompanied by the reduction of 1 equivalent of NAD$\sp+$ to NADH. Denaturation experiments suggested the formation of a covalent bond between the enzyme and one equivalent of adenosine. However, peptide mapping of the reduced enzyme-adenosine complex failed.Item Synthesis and Applications of Dirhodium Metallopeptides(2012-09-05) Zaykov, Alexander; Ball, Zachary T.; Parry, Ronald J.; Silberg, Jonathan J.The work describes the development of a new class of synthetic metallopeptides that features a dirhodium metal center. Combination of peptide and dirhodium properties leads to unique effects on peptide structure, peptide-protein interactions, and metal catalytic activity aimed at small molecule as well as protein substrates. Dirhodium is directly bound to carboxylate side chains of aspartate or glutamate yielding kinetically inert coordination complexes. This improves stability, allows purification and provides enhanced biocompatibility. Bridging of two side chains in the same sequence enables control of the peptide secondary structure. Dirhodium metallopeptides are applied to regulate coiled coil dimerization, stabilize and induce helical secondary structure, catalyze enantioselective organometallic transformation, and serve as ligands for proteins. These results lead to the development of hybrid organic-inorganic therapeutic agents, biological probes for study of protein-protein interactions, and enantioselective metallopeptide catalysis.Item The Evolutionary Ecology of Stereoisomeric Sesquiterpene Lactones in Xanthium strumarium(2012-11-30) Ahern, Jeffrey; Whitney, Kenneth D.; Rudgers, Jennifer A.; Parry, Ronald J.; Siemann, EvanThe ecological factors that maintain defensive chemical variation within and between plant species have intrigued ecologists for decades. While theory posits that polymorphisms may be maintained different forms of balancing selection, relatively few expe