Browsing by Author "Summers, Ryan M."
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Item Caffeine and Theophylline Inhibit beta-Galactosidase Activity and Reduce Expression in Escherichia coli(American Chemical Society, 2020) Horne, Jesse; Beddingfield, Elizabeth; Knapp, Madison; Mitchell, Stephanie; Crawford, Logan; Mills, Shelby Brooks; Wrist, Alexandra; Zhang, Shuyuan; Summers, Ryan M.; University of Alabama TuscaloosaThe beta-galactosidase enzyme is a common reporter enzyme that has been used extensively in microbiological and synthetic biology research. Here, we demonstrate that caffeine and theophylline, common natural methylxanthine products found in many foods and pharmaceuticals, negatively impact both the expression and activity of beta-galactosidase in Escherichia coli. The beta-galactosidase activity in E. coli grown with increasing concentrations of caffeine and theophylline was reduced over sixfold in a dose-dependent manner. We also observed decreasing lacZ mRNA transcript levels with increasing methylxanthine concentrations in the growth media. Similarly, caffeine and theophylline inhibit the activity of the purified beta-galactosidase enzyme, with an approximately 1.7-fold increase in K-M toward o-nitrophenyl-beta-galactoside and a concomitant decrease in nu(max). The authors recommend the use of alternative reporter systems when such methylxanthines are expected to be present.Item Cultivation and Genome Sequencing of Bacteria Isolated From the Coffee Berry Borer (Hypothenemus hampei), With Emphasis on the Role of Caffeine Degradation(Frontiers, 2021) Vega, Fernando E.; Emche, Sarah; Shao, Jonathan; Simpkins, Ann; Summers, Ryan M.; Mock, Meredith B.; Ebert, Dieter; Infante, Francisco; Aoki, Sayaka; Maul, Jude E.; United States Department of Agriculture (USDA); University of Alabama Tuscaloosa; University of Basel; El Colegio de la Frontera Sur (ECOSUR); University of Hawaii ManoaThe coffee berry borer, the most economically important insect pest of coffee worldwide, is the only insect capable of feeding and reproducing solely on the coffee seed, a food source containing the purine alkaloid caffeine. Twenty-one bacterial species associated with coffee berry borers from Hawai'i, Mexico, or a laboratory colony in Maryland (Acinetobacter sp. S40, S54, S55, Bacillus aryabhattai, Delftia lacustris, Erwinia sp. S38, S43, S63, Klebsiella oxytoca, Ochrobactrum sp. S45, S46, Pantoea sp. S61, Pseudomonas aeruginosa, P. parafulva, and Pseudomonas sp. S30, S31, S32, S37, S44, S60, S75) were found to have at least one of five caffeine N-demethylation genes (ndmA, ndmB, ndmC, ndmD, ndmE), with Pseudomonas spp. S31, S32, S37, S60 and P. parafulva having the full complement of these genes. Some of the bacteria carrying the ndm genes were detected in eggs, suggesting possible vertical transmission, while presence of caffeine-degrading bacteria in frass, e.g., P. parafulva (ndmABCDE) and Bacillus aryabhattai (ndmA) could result in horizontal transmission to all insect life stages. Thirty-five bacterial species associated with the insect (Acinetobacter sp. S40, S54, S55, B. aryabhattai, B. cereus group, Bacillus sp. S29, S70, S71, S72, S73, D. lacustris, Erwinia sp. S38, S43, S59, S63, K. oxytoca, Kosakonia cowanii, Ochrobactrum sp. S45, S46, Paenibacillus sp. S28, Pantoea sp. S61, S62, P. aeruginosa, P. parafulva, Pseudomonas sp. S30, S31, S32, S37, S44, S60, S75, Stenotrophomonas sp. S39, S41, S48, S49) might contribute to caffeine breakdown using the C-8 oxidation pathway, based on presence of genes required for this pathway. It is possible that caffeine-degrading bacteria associated with the coffee berry borer originated as epiphytes and endophytes in the coffee plant microbiota.Item Direct conversion of theophylline to 3-methylxanthine by metabolically engineered E-coli(BMC, 2015) Algharrawi, Khalid H. R.; Summers, Ryan M.; Gopishetty, Sridhar; Subramanian, Mani; University of Iowa; University of Alabama Tuscaloosa; University of BaghdadBackground: Methylxanthines are natural and synthetic compounds found in many foods, drinks, pharmaceuticals, and cosmetics. Aside from caffeine, production of many methylxanthines is currently performed by chemical synthesis. This process utilizes many chemicals, multiple reactions, and different reaction conditions, making it complicated, environmentally dissatisfactory, and expensive, especially for monomethylxanthines and paraxanthine. A microbial platform could provide an economical, environmentally friendly approach to produce these chemicals in large quantities. The recently discovered genes in our laboratory from Pseudomonas putida, ndmA, ndmB, and ndmD, provide an excellent starting point for precisely engineering Escherichia coli with various gene combinations to produce specific high-value paraxanthine and 1-, 3-, and 7-methylxanthines from any of the economical feedstocks including caffeine, theobromine or theophylline. Here, we show the first example of direct conversion of theophylline to 3-methylxanthine by a metabolically engineered strain of E. coli. Results: Here we report the construction of E. coli strains with ndmA and ndmD, capable of producing 3-methylxanthine from exogenously fed theophylline. The strains were engineered with various dosages of the ndmA and ndmD genes, screened, and the best strain was selected for large-scale conversion of theophylline to 3-methylxanthine. Strain pDdA grown in super broth was the most efficient strain; 15 mg/mL cells produced 135 mg/L (0.81 mM) 3-methylxanthine from 1 mM theophylline. An additional 21.6 mg/L (0.13 mM) 1-methylxanthine were also produced, attributed to slight activity of NdmA at the N-3-position of theophylline. The 1- and 3-methylxanthine products were separated by preparative chromatography with less than 5 % loss during purification and were identical to commercially available standards. Purity of the isolated 3-methylxanthine was comparable to a commercially available standard, with no contaminant peaks as observed by liquid chromatography-mass spectrophotometry or nuclear magnetic resonance. Conclusions: We were able to biologically produce and separate 100 mg of highly pure 3-methylxanthine from theophylline (1,3-dimethylxanthine). The N-demethylation reaction was catalyzed by E. coli engineered with N-demethylase genes, ndmA and ndmD. This microbial conversion represents a first step to develop a new biological platform for the production of methylxanthines from economical feedstocks such as caffeine, theobromine, and theophylline.Item Draft Genome Sequence of Pseudomonas sp. Strain CES, Containing the Entire Alkylxanthine Gene Cluster for Caffeine Breakdown(American Society of Microbiology, 2020) Summers, Ryan M.; Shao, Jonathan; Mock, Meredith B.; Yu, Chi Li; Vega, Fernando E.; University of Alabama Tuscaloosa; United States Department of Agriculture (USDA); University of IowaPseudomonas strain CES was isolated from caffeine-enriched soil and found to possess the N-demethylation pathway for caffeine breakdown. We report the nucleotide sequence of the draft genome with 5,827,822 bp, 62.6% G+C content, and 5,427 protein-coding regions.Item Elucidating the Basis for Substrate-Dependent Control of [4Fe4S] Redox Properties in Human DNA Primase(University of Alabama Libraries, 2024) Petersen, Courtney; Thompson, Matthew K.In eukaryotes, the fundamental tasks of DNA replication, processing, and repair are carried out via the coordinated action of several functionally diverse multiprotein machines. An increasing number of these proteins have been found to coordinate essential high potential [4Fe4S] clusters, though the exact roles these cofactors play in many of these systems remains poorly defined. Still, there is an innate need to understand the roles [4Fe4S] clusters play in these life-sustaining processes, as defects in [4Fe4S]-coordinating domains have been linked to cancer and other diseases of genomic instability. To date, most of the information available regarding the functional roles [4Fe4S] clusters play in eukaryotic DNA replication stem from studies involving DNA primase. During replication, DNA primase synthesizes short ribonucleotide primers that, after further modification, are used for processive DNA replication by the canonical polymerases δ and ϵ. Electrochemical studies conducted with human DNA primase have demonstrated DNA binding in the presence of ribonucleotide triphosphates (rNTPs) activates the [4Fe4S] cluster towards reversible redox activity. This observation implies rNTP binding 25 Å away allosterically modulates the redox properties of the [4Fe4S] cluster, however, the structural basis for this effect has not yet been explored. The purpose of this dissertation is to investigate the root cause of this behavior using a combination of spectroscopic approaches. Chapter 2 describes the results of chemical titration experiments in which oxidation of the [4Fe4S] cluster in p58C was monitored by Electron Paramagnetic Resonance spectroscopy. Chapter 3 then discusses the results of resonance Raman, Nuclear Resonance Vibrational Spectroscopy, and Fe K-edge Extended X-ray Absorption Fine Structure experiments focused on investigating the influence of substrate binding on cluster structure. Taken together, the results presented herein suggest the substrate-dependent redox behavior of the [4Fe4S] cluster is not exclusively caused by changes to cluster geometry and that other factors may be present that affect the immediate environment surrounding the cluster. Given the 25 Å distance between the [4Fe4S] and DNA binding sites, the work presented in this dissertation implicates the 5'-triphosphate of the initiating rNTP and several protein residues in an allosteric mechanism responsible for the communication of redox state information.Item Genetic characterization of caffeine degradation by bacteria and its potential applications(Wiley, 2015) Summers, Ryan M.; Mohanty, Sujit K.; Gopishetty, Sridhar; Subramanian, Mani; University of Alabama Tuscaloosa; University of IowaThe ability of bacteria to grow on caffeine as sole carbon and nitrogen source has been known for over 40 years. Extensive research into this subject has revealed two distinct pathways, N-demethylation and C-8 oxidation, for bacterial caffeine degradation. However, the enzymological and genetic basis for bacterial caffeine degradation has only recently been discovered. This review article discusses the recent discoveries of the genes responsible for both N-demethylation and C-8 oxidation. All of the genes for the N-demethylation pathway, encoding enzymes in the Rieske oxygenase family, reside on 13.2-kb genomic DNA fragment found in Pseudomonas putidaCBB5. A nearly identical DNA fragment, with homologous genes in similar orientation, is found in Pseudomonas sp. CES. Similarly, genes for C-8 oxidation of caffeine have been located on a 25.2-kb genomic DNA fragment of Pseudomonas sp. CBB1. The C-8 oxidation genes encode enzymes similar to those found in the uric acid metabolic pathway of Klebsiella pneumoniae. Various biotechnological applications of these genes responsible for bacterial caffeine degradation, including bio-decaffeination, remediation of caffeine-contaminated environments, production of chemical and fuels and development of diagnostic tests have also been demonstrated.Item Mixed culture biocatalytic production of the high-value biochemical 7-methylxanthine(BMC, 2023) Mock, Meredith B.; Summers, Ryan M.; University of Alabama TuscaloosaBackground 7-Methylxanthine, a derivative of caffeine noted for its lack of toxicity and ability to treat and even prevent myopia progression, is a high-value biochemical with limited natural availability. Attempts to produce 7-methylxanthine through purely chemical methods of synthesis are faced with complicated chemical processes and/or the requirement of a variety of hazardous chemicals, resulting in low yields and racemic mixtures of products. In recent years, we have developed engineered microbial cells to produce several methylxanthines, including 3-methylxanthine, theobromine, and paraxanthine. The purpose of this study is to establish a more efficient biosynthetic process for the production of 7-methylxanthine from caffeine.Results Here, we describe the use of a mixed-culture system composed of Escherichia coli strains engineered as caffeine and theobromine "specialist " cells. Optimal reaction conditions for the maximal conversion of caffeine to 7-methylxanthine were determined to be equal concentrations of caffeine and theobromine specialist cells at an optical density (600 nm) of 50 reacted with 2.5 mM caffeine for 5 h. When scaled-up to 560 mL, the simple biocatalytic reaction produced 183.81 mg 7-methylxanthine from 238.38 mg caffeine under ambient conditions, an 85.6% molar conversion. Following HPLC purification and solvent evaporation, 153.3 mg of dried 7-methylxanthine powder was collected, resulting in an 83.4% product recovery.Conclusion We present the first report of a biocatalytic process designed specifically for the production and purification of the high-value biochemical 7-methylxanthine from caffeine using a mixed culture of E. coli strains. This process constitutes the most efficient method for the production of 7-methylxanthine from caffeine to date.Item Production, modification, and characterization of natural bamboo fiber(University of Alabama Libraries, 2019) Rocky, AMK Bahrum Prang; Thompson, Amanda J.; University of Alabama TuscaloosaIn an effort to extract natural bamboo fiber (NBF) from bamboo for textiles and other uses, four bamboo species Bissetii, Giant Gray, Moso, and Red Margin were chosen for investigation. Conventional fibers such as cotton, polyester, regular rayon, and 12 commercial bamboo viscose were included for comparative study. By using different chemicals and routes, 144 types of NBFs were produced. Assessments on fiber yield percentages (40-77%), average lengths (1.50-37.10 cm), fineness (9.68—93.3 Tex), and overall qualities, determined at least 47 sets were prospective for commercial use. Hand-spinning was executed on three sets of NBFs after blending with cotton fibers. Investigation on moisture regain (M_R) and moisture content (M_C), revealed that bamboo plants and NBFs had M_R=8.0% and M_R=7.5% which was lower than rayon and bamboo viscose fiber (~11% and ~10%) but higher than raw cotton fibers (~5.7% and 5.4%). Among tensile properties, breaking tenacity of longer NBFs (33-37 cm) was 64-140 N/Tex and elongation at break was 2.0-2.5%; these values were 1.50-2.50 N/Tex and 8.0-11.0% respectively for blended yarns. Elemental, chemical, and crystallographic investigations were accomplished by energy-dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), ATR-FTIR (Attenuated Total Reflectance Fourier Transform Infra-Red), Raman spectroscopy (RS) and X-ray diffraction (XRD) techniques. Bamboo plants were estimated to be 70-78% carbon (C) and 20-30% oxygen (O) atoms, and O/C ratio of 0.26-0.33. The NBFs had a higher O/C ratio of 0.58-0.70. Comparisons of the spectra revealed the differences between bamboo-NBF and other fibers. Some distinct lignocellulosic peaks were in NBFs that could be responsible for unique properties. The crystallinity index (CI) of bamboo plants was 63-67% but CI of NBFs was higher 69-73% with crystallite sizes of 35-39 Å (3.5-3.9 nm). Four reflection planes and other properties are also documented. A suitable antibacterial test method was modified for quantitative estimation of bacterial reduction. Results suggest that though 11 out of 12 bamboo viscose products failed to exhibit inhibition against the bacteria, most of the bamboo and NBF specimens successfully showed a bacterial reduction of 8-95% against Klebsiella pneumoniae and 3-50% against Staphylococcus aureus.Item The role of outer-sphere residues and substrate-binding at the 3-mercaptopropionic acid dioxygenase (3mdo) active site: a combined spectroscopic and computational investigation(University of Alabama Libraries, 2021) York, Nicholas James; Pierce, Brad S.; University of Alabama TuscaloosaThiol Dioxygenases (TDOs) are non-heme iron enzymes that catalyze the O2-dependent oxidation of thiol-bearing amino acid derivatives to their corresponding sulfinic acid. Recently, considerable work has been focused on this class of enzymes, as sulfur metabolite imbalances have been correlated with neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and motor neuron disease. The active site of this enzyme class is comprised of a mononuclear Fe(II) bound by three protein derived histidine residues. A conserved feature among structurally characterized TDOs is a sequence of serine, histidine, and tyrosine residues adjacent to the iron active site, deemed the SHY motif. By far, the most well studied TDO is mammalian cysteine dioxygenase (CDO) which oxidizes L-cysteine to cysteine sulfinic acid with high substrate-specificity. Unlike CDO, bacterial 3-mercaptopropionate dioxygenase from Azotobacter vinelandii (Av3MDO) is a promiscuous TDO that oxidizes a variety of thiol substrates. Given both enzymes show similar active site geometries, the drastic difference in substrate specificity is not well understood. This dissertation aims to address two unresolved topics among Av3MDO and TDOs in general. The first is the mode in which native substrate, 3MPA, binds to Av3MDO. Arguments have been made for both bidentate coordination through the substrate thiolate and carboxylate and monodentate through the thiolate only. Herein, crystallographic, spectroscopic, and computational studies are used to determine the mode of 3MPA coordination to the iron active site. Bidentate coordination was observed for a crystal structure of bound inhibitor, 3- hydroxypropionic acid, which was used as a basis for computational models of bidentate 3MPA coordination. These models showed agreement with spectroscopic data using either nitric oxide or cyanide as spectroscopic probes. The second aspect investigated is the role of the SHY motif in the active site. For Av3MDO, alterations to the SHY motif have been shown to drastically attenuate activity (kcat), catalytic efficiency (kcat/KM), and formation of NO-bound ES complex. However, the interactions between the SHY motif and iron active site are not fully understood. Spectroscopic studies presented herein reveal the flexible hydrogen bonding capabilities of Tyr159 in the SHY motif and its direct influence on the electronic structure of the iron active site.Item Structural investigations and determination of biocatalyst potential of Pseudomonas putida CBB5(University of Alabama Libraries, 2020) Mills, Shelby Brooks; Summers, Ryan M.; University of Alabama TuscaloosaPseudomonas putida CBB5 has evolved the ability to metabolize caffeine and other methylxanthines to xanthine using a set of five enzymes, NdmABCDE. NdmABC are N1-, N3-, and N7-specific N-demethylases, respectively, that are part of the multicomponent Rieske oxygenase family. Structural investigations of NdmA, NdmB, and NdmD were conducted along with determining the applicability of harnessing this N-demethylation system for the bioproduciton of methylxanthines. Protein co-expression and purification of NdmA and NdmB confirmed the presence of an NdmAB complex that is constructed to perform N-demethylation. The interactions of NdmD and NdmAB were then elucidated because NdmD serves as the sole Rieske reductase for this set of enzymes and transfers electrons to each catalytic site. NdmD is unique amongst Rieske reductases because it contains an extra Rieske [2Fe-2S] cluster at the N-terminal end. The hypothesis is the Rieske [2Fe-2S] cluster on NdmD is used in conjunction with the Rieske-less NdmC enzyme and structural subunit NdmE to carry out the N7-demethylation of 7-methylxanthine to xanthine, but is not required for activity with the NdmA and NdmB enzymes. The results support the hypothesis and expand it by suggesting that the extra Rieske [2Fe-2S] cluster can be used as a secondary electron transport pathway to NdmAB. NdmA converts caffeine to theobromine, which is further N3-demethylated by NdmB, resulting in 7-methylxanthine. However, NdmA exhibits a slight promiscuity toward the N3-methyl group, resulting in 1.5% of caffeine being convertedto paraxanthine. Analysis of the NdmA and NdmB structures identified that only two of the nine amino acids in the binding pocket differ between NdmA and NdmB. Mutation of the two unique amino acids in NdmA to mimic the NdmB active site produced a mutant enzyme with a paraxanthine:theobromine ratio of at least 3:1, over a 100-fold improvement from the wild-type ratio (1:39). Additionally, a peptide loop near the active sites also differs between NdmA and NdmB. Mutation of the NdmA loop sequence to match that of the NdmB loop further increased the yield of paraxanthine. This research confirms that biocatalytic production of paraxanthine from caffeine is achievable and begins to optimize the starting reaction conditions.