Biosynthesis and evolution of anthracyclines

Natural products are a rich source of medication leads, accounting for about twothirds of all antibiotics and one-third of all anticancer drugs currently in use. Anthracyclines, which belong to the type II family of aromatic polyketides, are a medically essential class of natural products. They have one or more deoxysugar moieties attached at C–7 via O–glycosylation and are made up of the tetracyclic 7,8,9,10-tetrahydro-5,12-naphtacenoquinone aglycone chromophore. Anthracyclines are produced by soil-dwelling Gram-positive Actinobacteria. They are cytotoxic, but also cardiotoxic, which limits their clinical use. Minor changes in anthracyclines can reduce or eliminate their unwanted side-effects, while preserving their cytotoxicity. The literature review covers the biosynthesis of anthracyclines from the starting unit until the modification of the common aglycone scaffold. Furthermore, it depicts evolutionary events that have occurred during diversification of anthracycline biosynthetic pathways with special focus on the so-called tailoring steps. Finally, it explains the use of chimeragenesis as a protein engineering technique to trace the evolutionary events and to generate novel catalysts. This thesis focused on the biosynthesis of nogalamycin and elucidated the individual steps in the biosynthesis of L-rhodosamine using seven different enzymes. Furthermore, L-rhodosamine was attached to an anthracycline aglycone acceptor with a dual linkage system using the glycosyltransferase SnogD and the α- ketoglutarate–dependent monooxygenase SnoK. My work uncovered the 2''–hydroxylation function of the Rieske enzyme SnoT, which is a critical postmodification essential for nogalamycin bioactivity. This study also established the order of late-stage modification reactions with 2''–hydroxylation as the first step, followed by the 2–5'' carbocyclization and 4''–epimerization by SnoT, SnoK and SnoN, respectively. My thesis exploited chimeragenesis to uncover the evolutionary paths of anthracycline methyltransferases and α-ketoglutarate-dependent monooxygenases. The work elucidated how the 4'' epimerase SnoN has evolved via gene duplication and functional differentiation of the 2–5'' carbocyclase SnoK. In contrast, the 10– decarboxylase TamK and the 10–hydroxylase RdmB have evolved without a gene duplication event from the 4–O–methyltransferase DnrK. This study explains how the increase of anthracycline diversity may have occurred through subtle changes in the sequences of biosynthetic enzymes.Luonnonyhdisteet ovat lääkeaineaihioiden rikas lähde, joiden avulla on kehitetty noin kaksi kolmasosaa kliinisessä käytössä olevista antibiooteista ja kolmasosa syöpälääkkeistä. Antrasykliinit ovat lääkekehityksen kannalta tärkeä luonnonyhdisteiden luokka, mitkä kuuluvat tyypin II aromaattisiin polyketideihin. Antrasykliinejä tuottavat maaperässä elävät Gram-positiiviset Aktinobakteerit. Yhdisteet ovat sytotoksisia, mutta myös sydäntoksisia, mikä rajoittaa niiden kliinistä käyttöä. Pienet muutokset antrasykliinien rakenteissä voivat kuitenkin vähentää tai poistaa eitoivottuja haittavaikutuksia, samalla kuitenkin säilyttäen sytotoksisuuden. Kirjallisuuskatsaus kuvaa antrasykliinien biosynteesin yhteisen antrasykliinihiilirungon muodostumiseen saakka. Tämän lisäksi tutkielma kuvaa antrasykliinien evoluutiota ja monimuotoisuuden syntymistä keskittyen erityisesti niin sanottuihin muokkausreaktioihin. Lopuksi tutkielmassa käydään läpi kuinka proteiinien muokkaukseen käytettävää kimerageneesi-menetelmää voidaan käyttää proteiinievoluutiossa tapahtuvien muutosten tutkimiseen ja uusien proteiinikatalyyttien kehittämiseen. Tutkielmani keskittyy nogalamysiinin biosynteesiin, missä selvitin kuinka Lrhodosamiini sokeri muodostuun seitsemän entsyymin toimesta. Tämän lisäksi liitimme L-rhodosamiinin antrasykliiniin kaksinkertaisella sidoksella käyttäen glykosyylitransferaasia SnogD ja α-ketoglutaraattista riippuvaista oksygenaasia SnoK. Työni paljasti, että nogalamysiinin bioaktiivisuuden kannalta kriittisen 2'' hydroksylaatioryhmän asettaa paikalle Rieske entsyymi SnoT. Tutkielmani selvitti myös myöhäisen vaiheen muokkausreaktion järjestyksen, missä ensin tapahtuu SnoT proteiinin katalysoima 2'' hydroksylaatio, minkä jälkeen SnoK entsyymi katalysoi 2–5'' karbosyklisaatiota ja SnoN proteiini 4'' epimerisaatiota. Käytin kimerageneesi-menetelmää selvittääkseni eri antrasykliinien biosynteesiin liittyvien metyylitransferaasien ja α-ketoglutaraattista riippuvaisten oksygenaasien evoluutiota. Työni selvitti kuinka 4'' epimeraasi SnoN on muodostunut muinaisen 2–5'' karbosyklaasin SnoK tuotosta vastaavan geenin kahdentumisen ja funktionaalisen erilaistumisen seurauksena. Tämä on erilainen kuin mitä on tapahtunut 10–dekarboksylaasin TamK ja 10–hydroksylaasin RdmB kohdalla, missä proteiinien evoluution on tapahtunut 4–O–metyylitransferaasi DnrK proteiinista ilman geenien kahdentumista. Työni kuvaa kuinka hyvin pienet muutokset biosynteesistä vastuussa olevissa proteiineissa voivat johtaa antrasykliinien monimuotoisuuden kasvuun

The Rieske Oxygenase SnoT Catalyzes 2''-Hydroxylation of L-Rhodosamine in Nogalamycin Biosynthesis

Nogalamycin is an anthracycline anti-cancer agent that intercalates into the DNA double helix. The binding is facilitated by two carbohydrate units, L-nogalose and L-nogalamine, that interact with the minor and major grooves of DNA, respectively. However, recent investigations have shown that nogalamycin biosynthesis proceeds through the attachment of l-rhodosamine (2′′-deoxy-4′′-epi-L-nogalamine) to the aglycone. Herein, we demonstrate that the Rieske enzyme SnoT catalyzes 2′′-hydroxylation of L-rhodosamine as an initial post-glycosylation step. Furthermore, we establish that the reaction order continues with 2–5′′ carbocyclization and 4′′ epimerization by the non-heme iron and 2-oxoglutarate-dependent enzymes SnoK and SnoN, respectively. These late-stage tailoring steps are important for the bioactivity of nogalamycin due to involvement of the 2′′- and 4′′-hydroxy groups of ᴸ-nogalamine in hydrogen bonding interactions with DNA.</p

Enzymatic Synthesis of the C-glycosidic Moiety of Nogalamycin R

Carbohydrate moieties are essential for the biological activity of anthracycline anticancer agents such as nogalamycin, which contains l-nogalose and l-nogalamine units. The former of these is attached through a canonical O-glycosidic linkage, but the latter is connected via an unusual dual linkage composed of C–C and O-glycosidic bonds. In this work, we have utilized enzyme immobilization techniques and synthesized l-rhodosamine-thymidine diphosphate (TDP) from α-d-glucose-1-TDP using seven enzymes. In a second step, we assembled the dual linkage system by attaching the aminosugar to an anthracycline aglycone acceptor using the glycosyl transferase SnogD and the α-ketoglutarate dependent oxygenase SnoK. Furthermore, our work indicates that the auxiliary P450-type protein SnogN facilitating glycosylation is surprisingly associated with attachment of the neutral sugar l-nogalose rather than the aminosugar l-nogalamine in nogalamycin biosynthesis.</p

Evolution‐Guided Engineering of Non‐Heme Iron Enzymes Involved in Nogalamycin Biosynthesis

Microbes are competent chemists that are able to generate thousands of chemically complex natural products with potent biological activities. Key to the formation of this chemical diversity has been the rapid evolution of secondary metabolism. Many enzymes residing on these metabolic pathways have acquired atypical catalytic properties in comparison to their counterparts found in primary metabolism. The biosynthetic pathway of the anthracycline nogalamycin contains two such proteins, SnoK and SnoN, belonging to non‐heme iron and 2‐oxoglutarate‐dependent mono‐oxygenases. In spite of structural similarity, the two proteins catalyse distinct chemical reactions; SnoK is a C2–C5′′ carbocyclase, whereas SnoN catalyses stereoinversion at the adjacent C4′′ position. Here we have identified four structural regions involved in the functional differentiation and generated 30 chimeric enzymes to probe catalysis. Our analyses indicate that the carbocyclase SnoK is the ancestral form of the enzyme from which SnoN has evolved to catalyse stereoinversion at the neighboring carbon. The critical step in the appearance of epimerization activity has likely been the insertion of three residues near the C‐terminus, which allow repositioning of the substrate in front of the iron center. The loss of the original carbocyclization activity has then occurred with changes in four amino acids near the iron center that prohibit alignment of the substrate for formation of the C2–C5′′ bond. Our study provides detailed insights into the evolutionary processes that have enabled Streptomyces soil bacteria to become the major source of antibiotics and antiproliferative agents.</div

Diverse Combinatorial Biosynthesis Strategies for C–H Functionalization of Anthracyclinones

Streptomyces spp. are “nature’s antibiotic factories” that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in Streptomyces coelicolor M1152ΔmatAB that could produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues via two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and S. coelicolor M1152ΔmatAB expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant Streptomyces spp. hosts