Graduation date: 1994

The continued propagation of antibiotic resistance requires the development of new therapeutics. The lipopeptide antibiotic enduracidin has demonstrated high activity against Gram-positive pathogens including methicillin-resistant Staphylococcus aureus. In addition to a lack of cross-resistance with existing antibiotic classes, enduracidin has no known transferrable resistance mechanism. The development of enduracidin as a human therapeutic is hampered by its poor solubility in plasma. Utilizing chemical and genetic techniques, analogs of enduracidin have been produced and evaluated for biological activity. Making use of the hydroxyphenylglycine (Hpg) biosynthetic pathway, fluorine was incorporated into enduracidin with minimal to no loss of bioactivity. The semisynthetic chemical modification of enduracidin proved to be challenging. The chemical nitration of the Hpg residues was unsuccessful. Modifications to the lipid tail by cleavage at the C2-olefin with ozone and the use of Diels-Alder reagents to react with the lipid tail diene also proved unsuccessful. However, the reduction and dihydroxylation modifications of the lipid tail diene were successful. Introduction of polar hydroxyl groups onto the alkyl tail reduced bioactivity while reduction of the diene had no significant effect. Analysis of the biosynthetic pathways involved in producing the lipid tail and the unusual amino acid enduracididine yielded some insights into the formation of the antibiotic. Through complementation of mutants having disruptions in the biosynthetic gene cluster and crystallographic data, the function of EndR as a cyclase was established. Additionally, the use of 4-hydroxyarginine as an intermediate in enduracididine biosynthesis was demonstrated. The ability of EndQ to function as a transaminase on both 4-hydroxyarginine and 2-ketoenduracididine was also established. The specific functions of EndP and EndQ have not been determined. The introduction of the lipid tail diene by the three enzymes Orf39, Orf44 and Orf45 was confirmed. Orf45 functions as a CoA ligase and a dehydrogenase to introduce the C2 double bond. The functions of Orf39 and Orf44 appear to be the introduction of the C4 double bond and isomerization of the C2 olefin. / Graduation date: 2013

No description available.

No description available.

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

An alpha-amino ester hydrolase (AEH) applicable to synthesis of semi-synthetic antibiotics was cloned from the genomic DNA of Xanthomonas campestris pv. campestris sp. strain ATCC 33913. AEHs catalyze the synthesis and hydrolysis of alpha-amino beta-lactam antibiotics. The enzyme was characterized for thermodynamic and kinetic parameters. The enzyme shows optimal ampicillin hydrolytic activity at 25C and pH 6.8. The AEH enzymes have been shown to have excellent synthetic capability. Additionally, we demonstrated the first fully aqueous enzymatic one-pot synthesis of ampicillin direct from the natural product penicillin G eliminating the isolation of the intermediate 6-APA. Lastly, to improve the thermostability of the AEH a modified structure-guided consensus model of seven homologous enzymes was generated along with analysis of the B-factors from the available crystal structures of the known AEH from Xanthomonas citri. Our best variant, which is a quadruple mutant, E143H/A275P/N186D/V622I, which has a T_50_30, the temperature at which the half-life is 30 minutes, of 34C and 1.3-fold activity compared to wild-type. Overall, we have successfully improved the understanding of the AEH class of enzymes and applied a novel cascade application, demonstrating AEHs unique applicability in the synthesis of beta-lactam antibiotics. The improved thermostability will further improve the industrial relevance of AEHs.

No description available.

Among the myriad of naturally occurring bioactive compounds are the aminocyclopentitol-containing natural products that represent a family of sugar-derived microbial secondary metabolites, such as the antibiotics pactamycin, allosamidin, and trehazolin. Pactamycin, a structurally unique aminocyclitol antibiotic isolated from Streptomyces pactum, consists of a 5-membered ring aminocyclitol (cyclopentitol) unit, two aromatic rings (6-methylsalicylic acid (6-MSA) and 1-(3-Amino-phenyl)-ethanone or 3-aminoacetophenone) and a 1,1-dimethylurea. It has pronounced antibacterial, antitumor, antiviral, and antiplasmodial activities, but its development as a clinical drug was hampered by its broad cytotoxicity. Efforts to modulate its pharmacological and toxicity properties by structural modifications using synthetic organic chemistry have been difficult due to the complexity of its chemical structure. As part of our ongoing studies on the biosynthesis of aminocyclitol-derived bioactive natural products, we have identified the biosynthetic gene cluster of pactamycin in S. pactum ATCC 27456, which paves the way for a better understanding of pactamycin biosynthesis and generating novel pactamycin analogs through biosynthetic engineering. Through gene inactivations, feeding experiments, and in vitro enzymatic assay, we studied the biosynthesis of pactamycin, which include the modes of formation of the unique cyclopentitol unit, the 3-aminoacetophenone and the 6-methyl salicylic acid moieties. Armed with the tools needed to genetically engineer target strains of S. pactum, we were able to produce novel analogs of this untapped-class of natural products. TM-026 was generated from a ΔptmH (a radical SAM C-methyltransferase gene) mutant, whereas TM-025 was generated from a ΔptmH/ΔptmQ (a polyketide synthase gene) double knockout mutant. Both compounds show potent antimalarial activity, but lack significant antibacterial activity, and are about 10-30 times less toxic than pactamycin toward mammalian cells. The results suggest that distinct ribosomal binding selectivity or new mechanism(s) of action may be involved in their plasmodial growth inhibition, which may lead to the discovery of new antimalarial drugs and identification of new molecular targets within malarial parasites. TM-035 was also isolated from a ΔptmH mutant. However, we found that TM-035 showed no activity against bacteria, malarial parasites, and most tested mammalian cells, but it has potent growth inhibitory activity against two well-established human head and neck squamous cell carcinomas (SCC025 and SCC104) (IC₅₀ 725 nM) in an in vitro assay. More intriguingly, the compound is significantly less active against human primary epidermal keratinocytes (HPEK), demonstrating an interesting biological phenomenon and outstanding cell type selectivity, which may lead to the development of new anticancer chemotherapy. The production yield of pactamycin and its congeners under laboratory conditions is relatively low. This has hampered both mechanistic and preclinical studies of these promising compounds. To deepen our understanding of pactamycin biosynthesis and engineer mutant strains with improved production yields, we investigated pathway specific regulatory genes, ptmF and ptmE. Based on gene inactivation and RT-PCR studies, we found that the PtmF-PtmE system controls the transcription of the whole biosynthetic gene cluster. The results provide important insight into regulation of pactamycin biosynthesis and will contribute to future studies that aim at engineering high producing strains of S. pactum. / Graduation date: 2012 / Access restricted to the OSU Community at author's request from Feb. 28, 2012 - Feb. 28, 2013