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RISS 인기검색어
- 검색키워드
- 저자 : Ryan M. Summers (검색결과 2 건)
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Kim, Jun Hoe,Kim, Bong Heon,Brooks, Shelby,Kang, Seung Yeon,Summers, Ryan M.,Song, Hyun Kyu Elsevier 2019 Journal of molecular biology Vol.431 No.19
<P><B>Abstract</B></P> <P>Caffeine, found in many foods, beverages, and pharmaceuticals, is the most used chemical compound for mental alertness. It is originally a natural product of plants and exists widely in environmental soil. Some bacteria, such as <I>Pseudomonas putida</I> CBB5, utilize caffeine as a sole carbon and nitrogen source by degrading it through sequential <I>N</I>-demethylation catalyzed by five enzymes (NdmA, NdmB, NdmC, NdmD, and NdmE). The environmentally friendly enzymatic reaction products, methylxanthines, are high-value biochemicals that are used in the pharmaceutical and cosmetic industries. However, the structures and biochemical properties of bacterial <I>N</I>-demethylases remain largely unknown. Here, we report the structures of NdmA and NdmB, the initial <I>N</I> <SUB>1</SUB>- and <I>N</I> <SUB>3</SUB>-specific demethylases, respectively. Reverse-oriented substrate bindings were observed in the substrate-complexed structures, offering methyl position specificity for proper <I>N</I>-demethylation. For efficient sequential degradation of caffeine, these enzymes form a unique heterocomplex with 3:3 stoichiometry, which was confirmed by enzymatic assays, fluorescent labeling, and small-angle x-ray scattering. The binary structure of NdmA with the ferredoxin domain of NdmD, which is the first structural information for the plant-type ferredoxin domain in a complex state, was also determined to better understand electron transport during <I>N</I>-demethylation. These findings broaden our understanding of the caffeine degradation mechanism by bacterial enzymes and will enable their use for industrial applications.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Crystal structures of bacterial N-demethylase A and B were determined in apo and substrate-bound states. </LI> <LI> NdmAB forms a unique heterocomplex with 3:3 stoichiometry. </LI> <LI> Structure of the binary complex between NdmA and a ferredoxin domain of NdmD was determined. </LI> <LI> The structural and biochemical results provide a framework for engineering of caffeine degradation enzymes and thus increase the reaction products, methylxanthines, which are high-value biochemicals. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>
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Biocatalytic Production and Purification of the High-value Biochemical Paraxanthine
Meredith B. Mock,Shelby Brooks Mills,Ashley Cyrus,Hailey Campo,Tyler Dreischarf,Sydney Strock,Ryan M. Summers 한국생물공학회 2022 Biotechnology and Bioprocess Engineering Vol.27 No.4
Paraxanthine (1,7-dimethylxanthine), a purine alkaloid derivative of caffeine (1,3,7-trimethylxanthine), is a high-value biochemical with several applications in the pharmaceutical and cosmetic industries. However, chemical synthesis of paraxanthine requires harsh conditions and frequently results in low yield mixtures of non-specifically N-methylated compounds. We have recently demonstrated that the mutant bacterial N-demethylase NdmA4 with its partner reductase NdmD is capable of producing paraxanthine as the major metabolite from caffeine. Here, we report the construction and screening of several Escherichia coli strains to produce paraxanthine from caffeine by means of whole-cell biocatalysts using varying dosages of ndmA4, ndmD, and the frmAB formaldehyde dehydrogenase genes. Preliminary resting cell assay results with the best paraxanthine-producing strain, MBM019, showed a 33% molar conversion of caffeine, from 5 mM to 3.35 mM, resulting in approximately 0.90 mM paraxanthine. However, a small amount of 7-methylxanthine was unexpectedly produced at a concentration of approximately 0.35 mM. After optimizing reaction conditions to a cellular concentration of OD600 = 50 and a caffeine concentration of 5 mM, the reaction was scaled-up to a volume of 620 mL, producing 1.02 mM paraxanthine and consuming 2.49 mM caffeine. The purified paraxanthine was then isolated via preparatory scale chromatography, resulting in 104.1 mg of product at high purity. This is the first reported strain genetically optimized for the biosynthetic production of paraxanthine.
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