卡普拉霉素(Caprazamycin)是具有抑制細菌體內之磷酸-N-乙酰胞壁酰-五肽轉位酶 (MraY) 的核苷類抗生素的成員之一。雖然已有許多研究探討此抗生素之生物合成，但至今仍尚未完整。本研究進一步確定了數個參與卡普拉霉素生物合成之關鍵酵素的活性機轉，並針對兩種特殊非血基質的α-酮戊二酸依賴性酶 Cpz10 和 Cpz15進行蛋白質結構解析與作用機轉探討。在實驗結果中，Cpz15被證實其功能為將尿苷單磷酸轉化為5-尿苷醛的起始酶而非先前所認定的β-羥化酶。相反的，Cpz10則在此研究中被確認是β-羥化酶，能在體外試驗中將卡普拉霉素生合成之中間產物化合物 10羥基化為化合物 11。同時在剔除Cpz10基因片段之鍊黴菌發酵體內試驗中，我們發現到卡普拉霉素生合成之中間產物12的生成。此結果驗證了先前體外試驗的結果，β-羥化作用能驅使隨後的酰化反應來完成隨後的生物合成作用，並在爾後的環化作用扮演一關鍵酵素。在解析蛋白質結構的研究中，發現到中間產物化合物10在Cpz10的反應中心裡與活性胺基酸之pro-S氫原子形成六配位的構型來調控其立體選擇性與區域選擇性的反應作用。除此之外，Cpz10也同時招募了兩個鐵離子（Fe1 和 Fe2），其中Fe1 作為反應中心； Fe2 負責電荷轉移。此結果同時得到了 NBO 計算、 EPR 分析、迴聲動力學中的電荷流測試、氧化態判定與配位幾何分析的支持。本研究中結合了酵素生化測試、體內與體外生物活性測試與相關生物物理分析來促進我們對卡普拉霉素生物合成酵素的了解，同時也對於開發新一代此類型核苷類抗生素來對抗多重藥性病原體提供了一個更有效率的方法。

Caprazamycin is a member of nucleoside antibiotics inhibiting phospho-N-acetylmuramyl-pentapeptide translocase (MraY). The biosynthesis of nucleoside antibiotics has been studied to considerable extent but still far from completion. The present study determined the biochemical roles for selected enzymes involved in the biosynthesis of caprazamycin, particularly for two non-heme αKG-dependent enzymes Cpz10 and Cpz15. Cpz15 that was previously assigned as a β-hydroxylase is instead the starter enzyme converting uridine mono phosphate to uridine 5’ aldehyde. In contrast, Cpz10 is the actual β-hydroxylase hydroxylating synthetic compound 10 to compound 11 in vitro. Cpz10 was parallelly examined in vivo using the gene-deletion mutant Δcpz10, by which the major product collected is compound 12 agreeing with the in vitro result. The β-hydroxylation enables subsequent acylation toward full-blown biological activity and acts as a check point permitting the seven-membered ring formation. Crystal structures revealed that compound 10 is placed above the sixth coordinate with the pro-S hydrogen atom of β-carbon exposed to the sixth coordinate accounting for reaction regioselectivity/stereoselectivity. Beyond that, Cpz10 recruits two irons (Fe1 and Fe2): Fe1 serves as the reaction center; Fe2 is responsible for charge transfer. This finding is supported by NBO calculation and EPR analysis, echoing dynamic changes in charge flow, oxidation status, and coordination geometry during the Cpz10-mediated reaction. In conjunction with biochemical determination for other related enzymes, the in vitro, in vivo and biophysical profiling advance our understanding of caprazamycin biosynthesis, which is conducive to pathway engineering to generate more effective nucleoside antibiotics against multidrug resistant pathogens.

ACKNOWLEDGMENTS........................................................................................i
ABSTARCT..............................................................................................ii
TABLE OF CONTENTS......................................................................................v
LIST OF TABLES ......................................................................................vii
LIST OF FIGURES.....................................................................................viii
LIST OF SCHEMES.......................................................................................ix
LIST OF ABBREVIATIONS..................................................................................x
Chapter one: Introduction .............................................................................1
1.1. Natural Products – Significance ..................................................................1
1.2. Peptidoglycan cell wall...........................................................................2
1.3. MraY – Structure and Function.....................................................................4
1.4. Current understanding of the caprazamycin biosynthesis pathway....................................7
1.5. Non-heme enzymes in the biosynthesis pathway of nucleoside antibiotics............................8
Chapter two: Material and Methods.....................................................................11
2.1. General chemicals, reagents and analytical methods…..............................................11
2.2. Bacteria strains and plasmids used in this study.................................................12
2.3. Construction of pMKBAC02.........................................................................13
2.4. Isolation of entire caprazamycin aglycone biosynthetic gene cluster into pMKBAC..................14
2.5. Generation of cpz10-deficient mutants via in vitro CRISPR-Cas9 digestion.........................17
2.6. In vitro transcription of sgRNA was performed using the TranscriptAid............................17
2.7. Cloning for gene expression......................................................................18
2.8. Site-directed mutagenesis........................................................................20
2.9. Reactions with Cpz15 and Mur16...................................................................22
2.10. Activity of Mur17...............................................................................22
2.11. Reaction with cpz10.............................................................................23
2.12. Reactions with cpz27 and cpz12..................................................................24
2.13. Reaction with mur23.............................................................................24
2.14. Purification of cpz10 knockout intermediate.....................................................25
2.15. UV/Vis spectral analysis........................................................................25
2.16. EPR Spectroscopic Method........................................................................26
2.17. Crystallization and data collection.............................................................26
2.18. Structure determination and refinement..........................................................29
2.19. Computational methods...........................................................................29
Chapter three: Results and Discussion.................................................................31
3.1. Characterization of a non-heme enzyme that generates Uridine-5’-aldehyd..........................31
3.2. Cpz10 is the β-hydroxylase.......................................................................34
3.3. Cpz10 X-ray crystal structures reveal mechanism of hydroxylation by two iron ions................38
3.4. EPR study for Cpz10..............................................................................50
3.5. Investigation of the nature of the Fe (III) and Fe (II) EPR signals by using the NBO analysis....52
3.6. In vivo study of Cpz10 knock out by CRISPR/cas9..................................................58
3.7. Cpz27 is responsible for phosphorylation of compound 13..........................................64
3.8. Decarboxylation of compound 10 by Mur23..........................................................68
Chapter four. Conclusion..............................................................................72
References............................................................................................80
Appendixes............................................................................................86

1 Koehn, F. E. & Carter, G. T. The evolving role of natural products in drug discovery. Nature reviews Drug discovery 4, 206-220 (2005).
2 Harvey, A. L. Natural products in drug discovery. Drug discovery today 13, 894-901 (2008).
3 Li, J. W.-H. & Vederas, J. C. Drug discovery and natural products: end of an era or an endless frontier? Science 325, 161-165 (2009).
4 Davies, H. M. Synthetic lessons from nature. Nature 459, 786-787 (2009).
5 Walsh, C. T. & Fischbach, M. A. Natural products version 2.0: connecting genes to molecules. Journal of the American Chemical Society 132, 2469-2493 (2010).
6 Watve, M. G., Tickoo, R., Jog, M. M. & Bhole, B. D. How many antibiotics are produced by the genus Streptomyces? Archives of microbiology 176, 386-390 (2001).
7 Bérdy, J. Thoughts and facts about antibiotics: where we are now and where we are heading. The Journal of antibiotics 65, 385-395 (2012).
8 Bugg, T. & Walsh, C. Intracellular steps of bacterial cell wall peptidoglycan biosynthesis: enzymology, antibiotics, and antibiotic resistance. Natural product reports 9, 199-215 (1992).
9 Van Heijenoort, J. Recent advances in the formation of the bacterial peptidoglycan monomer unit. Natural product reports 18, 503-519 (2001).
10 Barton, D. & Meth-Cohn, O. Comprehensive natural products chemistry. (Newnes, 1999).
11 Park, J. T. & Johnson, M. J. Accumulation of labile phosphate in Staphylococcus aureus grown in the presence of penicillin. Journal of Biological Chemistry 179, 585-592 (1949).
12 Kimura, K.-i. & Bugg, T. D. Recent advances in antimicrobial nucleoside antibiotics targeting cell wall biosynthesis. Natural product reports 20, 252-273 (2003).
13 Winn, M., Goss, R. J., Kimura, K.-i. & Bugg, T. D. Antimicrobial nucleoside antibiotics targeting cell wall assembly: Recent advances in structure–function studies and nucleoside biosynthesis. Natural product reports 27, 279-304 (2010).
14 Lovering, A. L., Safadi, S. S. & Strynadka, N. C. Structural perspective of peptidoglycan biosynthesis and assembly. Annual review of biochemistry 81, 451-478 (2012).
15 Bouhss, A., Trunkfield, A. E., Bugg, T. D. & Mengin-Lecreulx, D. The biosynthesis of peptidoglycan lipid-linked intermediates. FEMS microbiology reviews 32, 208-233 (2007).
16 Sauvage, E., Kerff, F., Terrak, M., Ayala, J. A. & Charlier, P. The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis. FEMS microbiology reviews 32, 234-258 (2008).
17 Kahne, D., Leimkuhler, C., Lu, W. & Walsh, C. Glycopeptide and lipoglycopeptide antibiotics. Chemical reviews 105, 425-448 (2005).
18 Dong, Y. Y. et al. Structures of DPAGT1 explain glycosylation disease mechanisms and advance TB antibiotic design. Cell 175, 1045-1058. e1016 (2018).
19 Igarashi, M. et al. Caprazamycin B, a novel anti-tuberculosis antibiotic, from Streptomyces sp. The Journal of antibiotics 56, 580-583 (2003).
20 Struve, W. & Neuhaus, F. Evidence for an initial acceptor of UDP-NAc-muramyl-pentapeptide in the synthesis of bacterial mucopeptide. Biochemical and biophysical research communications 18, 6-12 (1965).
21 Anderson, J. S., Matsuhashi, M., Haskin, M. A. & Strominger, J. L. Lipid-phosphoacetylmuramyl-pentapeptide and lipid-phosphodisaccharide-pentapeptide: presumed membrane transport intermediates in cell wall synthesis. Proceedings of the National Academy of Sciences of the United States of America 53, 881 (1965).
22 Malek Zadeh, S. et al. Theoretical Study of Intermolecular Interactions between Critical Residues of Membrane Protein MraYAA and Promising Antibiotic Muraymycin D2. ACS omega 5, 22739-22749 (2020).
23 Lehrman, M. A. Commentary: A family of UDP-GlcNAc/MurNAc: polyisoprenol-P GlcNAc/MurNAc-1-P transferases. Glycobiology 4, 768-771 (1994).
24 Hakulinen, J. K. et al. MraY–antibiotic complex reveals details of tunicamycin mode of action. Nature chemical biology 13, 265-267 (2017).
25 Chung, B. C. et al. Structural insights into inhibition of lipid I production in bacterial cell wall synthesis. Nature 533, 557-560 (2016).
26 Wiegmann, D. et al. Muraymycin nucleoside-peptide antibiotics: uridine-derived natural products as lead structures for the development of novel antibacterial agents. Beilstein journal of organic chemistry 12, 769-795 (2016).
27 Mashalidis, E. H. et al. Chemical logic of MraY inhibition by antibacterial nucleoside natural products. Nature communications 10, 1-12 (2019).
28 McErlean, M., Liu, X., Cui, Z., Gust, B. & Van Lanen, S. Identification and characterization of enzymes involved in the biosynthesis of pyrimidine nucleoside antibiotics. Natural Product Reports (2021).
29 Draelos, M. M., Thanapipatsiri, A., Sucipto, H. & Yokoyama, K. Cryptic phosphorylation in nucleoside natural product biosynthesis. Nature Chemical Biology, 1-9 (2020).
30 Barnard-Britson, S. et al. Amalgamation of nucleosides and amino acids in antibiotic biosynthesis: discovery of an L-threonine: uridine-5′-aldehyde transaldolase. Journal of the American Chemical Society 134, 18514-18517 (2012).
31 Ushimaru, R. & Liu, H.-w. Biosynthetic origin of the atypical stereochemistry in the thioheptose core of albomycin nucleoside antibiotics. Journal of the American Chemical Society 141, 2211-2214 (2019).
32 Kaysser, L. et al. Formation and attachment of the deoxysugar moiety and assembly of the gene cluster for caprazamycin biosynthesis. Applied and environmental microbiology 76, 4008-4018 (2010).
33 Fujita, Y. et al. Muraminomicins, new lipo-nucleoside antibiotics from Streptosporangium sp. SANK 60501-structure elucidations of muraminomicins and supply of the core component for derivatization. The Journal of antibiotics 72, 943-955 (2019).
34 Ishizaki, Y. et al. Inhibition of the first step in synthesis of the mycobacterial cell wall core, catalyzed by the GlcNAc-1-phosphate transferase WecA, by the novel caprazamycin derivative CPZEN-45. Journal of Biological Chemistry 288, 30309-30319 (2013).
35 Shiraishi, T., Hiro, N., Igarashi, M., Nishiyama, M. & Kuzuyama, T. Biosynthesis of the antituberculous agent caprazamycin: Identification of caprazol-3ʺ-phosphate, an unprecedented caprazamycin-related metabolite. The Journal of general and applied microbiology 62, 164-166 (2016).
36 Tang, X. et al. A two-step sulfation in antibiotic biosynthesis requires a type III polyketide synthase. Nature chemical biology 9, 610-615 (2013).
37 Cui, Z. et al. Pyridoxal-5′-phosphate-dependent alkyl transfer in nucleoside antibiotic biosynthesis. Nature Chemical Biology, 1-8 (2020).
38 Yang, Z., Unrine, J., Nonaka, K. & Van Lanen, S. G. Fe (II)-dependent, uridine-5′-monophosphate α-ketoglutarate dioxygenases in the synthesis of 5′-modified nucleosides. Methods in enzymology 516, 153-168 (2012).
39 Cui, Z. et al. Enzymatic synthesis of the ribosylated glycyl-uridine disaccharide core of peptidyl nucleoside antibiotics. The Journal of organic chemistry 83, 7239-7249 (2018).
40 Kaysser, L. et al. Identification and manipulation of the caprazamycin gene cluster lead to new simplified liponucleoside antibiotics and give insights into the biosynthetic pathway. Journal of Biological Chemistry 284, 14987-14996 (2009).
41 Solomon, E. I., Goudarzi, S. & Sutherlin, K. D. O2 Activation by Non-Heme Iron Enzymes. Biochemistry 55, 6363-6374, doi:10.1021/acs.biochem.6b00635 (2016).
42 Goudarzi, S. et al. Evaluation of a concerted vs. sequential oxygen activation mechanism in α-ketoglutarate–dependent nonheme ferrous enzymes. Proceedings of the National Academy of Sciences 117, 5152-5159 (2020).
43 Solomon, E. I., Decker, A. & Lehnert, N. Non-heme iron enzymes: contrasts to heme catalysis. Proceedings of the National Academy of Sciences 100, 3589-3594 (2003).
44 Koketsu, K. et al. Refined regio-and stereoselective hydroxylation of L-pipecolic acid by protein engineering of L-proline cis-4-hydroxylase based on the X-ray crystal structure. ACS synthetic biology 4, 383-392 (2015).
45 O'Brien, J. R., Schuller, D. J., Yang, V. S., Dillard, B. D. & Lanzilotta, W. N. Substrate-induced conformational changes in Escherichia coli taurine/α-ketoglutarate dioxygenase and insight into the oligomeric structure. Biochemistry 42, 5547-5554 (2003).
46 Cui, Z. et al. Pyridoxal-5′-phosphate-dependent alkyl transfer in nucleoside antibiotic biosynthesis. Nature chemical biology 16, 904-911 (2020).
47 Horbal, L., Fedorenko, V. & Luzhetskyy, A. Novel and tightly regulated resorcinol and cumate-inducible expression systems for Streptomyces and other actinobacteria. Applied microbiology and biotechnology 98, 8641-8655 (2014).
48 Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F. & Hopwood, D. A. Practical streptomyces genetics. Vol. 291 (John Innes Foundation Norwich, 2000).
49 Jiang, W. & Zhu, T. F. Targeted isolation and cloning of 100-kb microbial genomic sequences by Cas9-assisted targeting of chromosome segments. nature protocols 11, 960-975 (2016).
50 Otwinowski, Z. & Minor, W. [20] Processing of X-ray diffraction data collected in oscillation mode. Methods in enzymology 276, 307-326 (1997).
51 McCoy, A. J. et al. Phaser crystallographic software. Journal of applied crystallography 40, 658-674 (2007).
52 Vagin, A. A. et al. REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. Acta Crystallographica Section D: Biological Crystallography 60, 2184-2195 (2004).
53 Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallographica Section D: Biological Crystallography 66, 486-501 (2010).
54 Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix. refine. Acta Crystallographica Section D: Biological Crystallography 68, 352-367 (2012).
55 DeLano, W. L. The PyMOL molecular graphics system. http://www. pymol. org (2002).
56 Reed, A. E., Curtiss, L. A. & Weinhold, F. Intermolecular interactions from a natural bond orbital, donor-acceptor viewpoint. Chemical Reviews 88, 899-926 (1988).
57 Weinhold, F. Discovering chemistry with natural bond orbitals. (John Wiley & Sons, 2012).
58 Wadt, W. & PJ, H. Ab initio effective core potentials formolecular calculations. Potentials for K to Au including theoutermost core orbitals. J Chem. Phys 82, 299-305 (1985).
59 Frisch, M. et al. Gaussian Inc. Wallingford Ct 2009 (2009).
60 Rajakovich, L. J. et al. A new microbial pathway for organophosphonate degradation catalyzed by two previously misannotated non-heme-iron oxygenases. Biochemistry 58, 1627-1647 (2019).
61 Yang, Z. et al. Characterization of LipL as a non-heme, Fe (II)-dependent α-ketoglutarate: UMP dioxygenase that generates uridine-5′-aldehyde during A-90289 biosynthesis. Journal of Biological Chemistry 286, 7885-7892 (2011).
62 Tars, K. et al. Targeting carnitine biosynthesis: discovery of new inhibitors against γ-butyrobetaine hydroxylase. Journal of medicinal chemistry 57, 2213-2236 (2014).
63 Scott, T. A., Heine, D., Qin, Z. & Wilkinson, B. An L-threonine transaldolase is required for L-threo-β-hydroxy-α-amino acid assembly during obafluorin biosynthesis. Nature communications 8, 1-11 (2017).
64 Yan, W. et al. Endoperoxide formation by an α-ketoglutarate-dependent mononuclear non-haem iron enzyme. Nature 527, 539-543 (2015).
65 Light, K. M., Hangasky, J. A., Knapp, M. J. & Solomon, E. I. Spectroscopic studies of the mononuclear non-heme FeII enzyme FIH: Second-sphere contributions to reactivity. Journal of the American Chemical Society 135, 9665-9674 (2013).
66 Elkins, J. M. et al. X-ray crystal structure of Escherichia coli taurine/α-ketoglutarate dioxygenase complexed to ferrous iron and substrates. Biochemistry 41, 5185-5192 (2002).
67 Shanmugam, M., Quareshy, M., Cameron, A. D., Bugg, T. D. & Chen, Y. Light‐Activated Electron Transfer and Catalytic Mechanism of Carnitine Oxidation by Rieske‐Type Oxygenase from Human Microbiota. Angewandte Chemie International Edition 60, 4529-4534 (2021).
68 Martinez, S. & Hausinger, R. P. Catalytic mechanisms of Fe (II)-and 2-oxoglutarate-dependent oxygenases. Journal of Biological Chemistry 290, 20702-20711 (2015).
69 Zhou, J. et al. Spectroscopic studies of substrate interactions with clavaminate synthase 2, a multifunctional α-KG-dependent non-heme iron enzyme: Correlation with mechanisms and reactivities. Journal of the American Chemical Society 123, 7388-7398 (2001).
70 Rabe, P., Kamps, J. J., Schofield, C. J. & Lohans, C. T. Roles of 2-oxoglutarate oxygenases and isopenicillin N synthase in β-lactam biosynthesis. Natural product reports 35, 735-756 (2018).
71 Schrödinger, L. The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC (2017). Google Scholar There is no corresponding record for this reference.
72 Ryle, M. J. et al. O2-and α-ketoglutarate-dependent tyrosyl radical formation in TauD, an α-keto acid-dependent non-heme iron dioxygenase. Biochemistry 42, 1854-1862 (2003).
73 Bradley, J. M., Svistunenko, D. A., Moore, G. R. & Le Brun, N. E. Tyr25, Tyr58 and Trp133 of Escherichia coli bacterioferritin transfer electrons between iron in the central cavity and the ferroxidase centre. Metallomics 9, 1421-1428 (2017).
74 Blasiak, L. C., Vaillancourt, F. H., Walsh, C. T. & Drennan, C. L. Crystal structure of the non-haem iron halogenase SyrB2 in syringomycin biosynthesis. Nature 440, 368-371 (2006).
75 Neugebauer, M. E. et al. A family of radical halogenases for the engineering of amino-acid-based products. Nature chemical biology 15, 1009-1016 (2019).
76 de Sousa, D. P. et al. Redox-and EPR-Active Graphene Diiron Complex Nanocomposite. Langmuir 35, 12339-12349 (2019).
77 Tampieri, F., Silvestrini, S., Riccò, R., Maggini, M. & Barbon, A. A comparative electron paramagnetic resonance study of expanded graphites and graphene. Journal of Materials Chemistry C 2, 8105-8112 (2014).
78 Bhattacharyya, A., Schmidt, M. P., Stavitski, E. & Martínez, C. E. Iron speciation in peats: chemical and spectroscopic evidence for the co-occurrence of ferric and ferrous iron in organic complexes and mineral precipitates. Organic Geochemistry 115, 124-137 (2018).
79 Weckhuysen, B. M., Heidler, R. & Schoonheydt, R. A. in Characterization I 295-335 (Springer, 2004).
80 Liberman, I. et al. Active-site modulation in an Fe-porphyrin-based metal–organic framework through ligand axial coordination: Accelerating electrocatalysis and charge-transport kinetics. Journal of the American Chemical Society 142, 1933-1940 (2020).
81 Iwamoto, L. S., Serra, O. A., Manso, C., Prado, M. & Iamamoto, Y. One pot obtention of a tetrabutylammonium hydroxide solution for ironporphyrin-OH-interaction studies in organic solvents. Química Nova 22, 277-279 (1999).
82 Hirst, J. et al. Replacement of the axial histidine ligand with imidazole in cytochrome c peroxidase. 2. Effects on heme coordination and function. Biochemistry 40, 1274-1283 (2001).
83 Wolfe, M. D. & Lipscomb, J. D. Hydrogen peroxide-coupled cis-diol formation catalyzed by naphthalene 1, 2-dioxygenase. Journal of Biological Chemistry 278, 829-835 (2003).
84 Bou-Abdallah, F. & Chasteen, N. D. Spin concentration measurements of high-spin (g′= 4.3) rhombic iron (III) ions in biological samples: theory and application. JBIC Journal of Biological Inorganic Chemistry 13, 15-24 (2008).
85 Wörsdörfer, B. et al. Organophosphonate-degrading PhnZ reveals an emerging family of HD domain mixed-valent diiron oxygenases. Proceedings of the National Academy of Sciences 110, 18874-18879 (2013).
86 Fufezan, C., Zhang, C., Krieger-Liszkay, A. & Rutherford, A. W. Secondary Quinone in Photosystem II of Thermosynechococcus elongatus: Semiquinone− Iron EPR Signals and Temperature Dependence of Electron Transfer. Biochemistry 44, 12780-12789 (2005).
87 Dong, M. et al. Organometallic and radical intermediates reveal mechanism of diphthamide biosynthesis. Science 359, 1247-1250 (2018).
88 Xing, G. et al. A coupled dinuclear iron cluster that is perturbed by substrate binding in myo-inositol oxygenase. Biochemistry 45, 5393-5401 (2006).
89 Doi, K., McCracken, J., Peisach, J. & Aisen, P. The binding of molybdate to uteroferrin. Hyperfine interactions of the binuclear center with 95Mo, 1H, and 2H. Journal of Biological Chemistry 263, 5757-5763 (1988).
90 Oloo, W. N. et al. Identification of a low-spin acylperoxoiron (III) intermediate in bio-inspired non-heme iron-catalysed oxidations. Nature communications 5, 1-9 (2014).
91 Alabugin, I. V., Manoharan, M., Peabody, S. & Weinhold, F. Electronic basis of improper hydrogen bonding: a subtle balance of hyperconjugation and rehybridization. Journal of the American Chemical Society 125, 5973-5987 (2003).
92 Alberti, F. & Corre, C. Editing streptomycete genomes in the CRISPR/Cas9 age. Natural product reports 36, 1237-1248 (2019).
93 Kallifidas, D., Jiang, G., Ding, Y. & Luesch, H. Rational engineering of Streptomyces albus J1074 for the overexpression of secondary metabolite gene clusters. Microbial cell factories 17, 1-14 (2018).
94 Ochi, K., Ezaki, M., Iwami, M., Komori, T. & Kohsaka, M. (Google Patents, 1990).
95 Kapp, U. et al. Structure of Deinococcus radiodurans tunicamycin-resistance protein (TmrD), a phosphotransferase. Acta Crystallographica Section F: Structural Biology and Crystallization Communications 64, 479-486 (2008).
96 Yang, Z. et al. Functional and kinetic analysis of the phosphotransferase CapP conferring selective self-resistance to capuramycin antibiotics. Journal of Biological Chemistry 285, 12899-12905 (2010).
97 Cui, Z. et al. Self-resistance during muraymycin biosynthesis: a complementary nucleotidyltransferase and phosphotransferase with identical modification sites and distinct temporal order. Antimicrobial agents and chemotherapy 62 (2018).
98 Jiang, L. et al. Identification of novel mureidomycin analogues via rational activation of a cryptic gene cluster in Streptomyces roseosporus NRRL 15998. Scientific reports 5, 1-13 (2015).
99 Wright, G. D. Opportunities for natural products in 21 st century antibiotic discovery. Natural product reports 34, 694-701 (2017).
100 Valegård, K. et al. The structural basis of cephalosporin formation in a mononuclear ferrous enzyme. Nature structural & molecular biology 11, 95-101 (2004).
101 Bjørnstad, L. G., Zoppellaro, G., Tomter, A. B., Falnes, P. Ø. & Andersson, K. K. Spectroscopic and magnetic studies of wild-type and mutant forms of the Fe (II)-and 2-oxoglutarate-dependent decarboxylase ALKBH4. Biochemical Journal 434, 391-398 (2011).
102 Chang, C. Y. et al. Biosynthesis of streptolidine involved two unexpected intermediates produced by a dihydroxylase and a cyclase through unusual mechanisms. Angewandte Chemie 126, 1974-1979 (2014).
103 Bethe, H. Termaufspaltung in kristallen. Annalen der Physik 395, 133-208 (1929).
104 Van Vleck, J. Theory of the variations in paramagnetic anisotropy among different salts of the iron group. Physical Review 41, 208 (1932).
105 Penney, W. G. & Schlapp, R. The influence of Crystalline Fields on the Susceptibilities of Salts of Paramagnetic Ions. I. The rare earths, especially Pr and Nd. Physical Review 41, 194 (1932).
106 Schlapp, R. & Penney, W. G. Influence of crystalline fields on the susceptibilities of salts of paramagnetic ions. II. The iron group, especially Ni, Cr and Co. Physical Review 42, 666 (1932).
107 Levine, I. N., Busch, D. H. & Shull, H. Quantum chemistry. Vol. 6 (Pearson Prentice Hall Upper Saddle River, NJ, 2009).
108 Baker, H. M., Anderson, B. F. & Baker, E. N. Dealing with iron: common structural principles in proteins that transport iron and heme. Proceedings of the National Academy of Sciences 100, 3579-3583 (2003).
109 von Liponukleosid-Antibiotika, B. Biosynthesis of liponucleoside antibiotics in Streptomyces: Molecular and biochemical investigations of the caprazamycin and the liposidomycin gene cluster.