Combinatorial Biosynthesis

DOI: 10.1002/anie.200603176

On the Acceptor Substrate of C-Glycosyl-transferase UrdGT2: Three PrejadomycinC-Glycosides from an Engineered Mutantof Streptomyces globisporus 1912DlndE(urdGT2)**

Irfan Baig, Madan Kharel, Anton Kobylyanskyy,Lili Zhu, Yuriy Rebets, Bohdan Ostash,Andriy Luzhetskyy, Andreas Bechthold,Victor A. Fedorenko,* and J$rgen Rohr*

The landomycins 1–4 produced by Streptomyces cyanogenusS-136 and S. globisporus 1912 are angucycline antibiotics witha strong activity against various cancer cell lines, in particular

[*] A. Kobylyanskyy,[+] Dr. Y. Rebets, Dr. B. Ostash,Prof. Dr. V. A. FedorenkoL’viv National UniversityDepartment of Genetic and BiotechnologyGrushevskyy St. 4, 79005 L’viv (Ukraine)E-mail: v_fedorenko@franko.lviv.ua

Dr. I. Baig,[+] Dr. M. Kharel,[+] Dr. L. Zhu,[+] Prof. Dr. J. RohrUniversity of KentuckyDepartment of Pharmaceutical Sciences, College of Pharmacy725 Rose Street, Lexington, KY 40536-0082 (USA)Fax: (+1)859-257-7564E-mail: jrohr2@email.uky.edu

Dr. A. Luzhetskyy, Prof. Dr. A. BechtholdAlbert-Ludwigs-UniversitGt FreiburgPharmazeutische BiologieStefan-Meier-Strasse 19, 79104 Freiburg (Germany)E-mail: andreas.bechthold@pharmazie.uni-freiburg.de

[+] These four authors contributed equally to this work.

[**] This work was supported financially by the U.S. National Institutesof Health (NIH grant CA 102102 to J.R.) and by the DAAD (DAADfellowship A/05/28943 to Y.R.). The University of Kentucky corefacilities for NMR spectroscopy and mass spectrometry areacknowledged for the use of their instruments and their service,respectively.

Supporting information for this article is available on the WWWunder http://www.angewandte.org or from the author.

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7842 � 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2006, 45, 7842 –7846

against prostate cancer cell lines.[1–6] The landomycins areclosely related to the urdamycins (e.g., urdamycin A (5)).[7–9]

Both possess a polyketide-derived angucyclinone core andsugar moieties consisting of d-olivose and l-rhodinose build-ing blocks. Major structural differences were found in theassembly of the sugar moieties and in the oxygenation patternof the polyketide core moiety.[1, 4,5, 10–12]

The availability of the biosynthetic gene clusters ofurdamycins and landomycins[7,13–17] made it possible to trackdown the genes encoding the biosynthetic enzymes respon-sible for the unique structural features of the two types ofcompounds, which are also responsible for the significantvariations in the biological activities of these related anti-cancer drugs.[18] The overall structural and biosyntheticsimilarity between these two closely related sets of antibioticsallowed several successful combinatorial, biosynthetic gene-combination experiments, leading to new hybrid mole-cules.[5,6, 15,19–24]

The most striking structural difference is that the trisac-charide chain found in the urdamycins is C-glycosidicallylinked at C9, while the oligosaccharide chains found in thedifferent landomycins are O-glycosidically linked at the 8-position. The enzymes responsible for the first glycosyltransfer step for these chains, the glycosyl transferases (GT)UrdGT2 and LanGT2/LndGT2, are closely related. LndGT2shows 53% amino acid identity (68% similarity) withUrdGT2. However, the acceptor substrates of these relatedGTs appear quite different, not only because one is a C-GTand the other an O-GT, but also with regard to the timing ofthe first GT step within the urdamycin and landomycin

biosyntheses. In landomycin biosynthesis, all but one oxygen-ation step in the aglycon formation appear to occur prior tothe first GT step,[5] while for the urdamycin biosynthesis thesequence of biosynthetic events (oxygen attachment at 12-and 12b-positions by UrdE and UrdM, respectively, before orafter the C-glycosylation step) remained the subject ofdebate, and the nature of the acceptor substrate of theimportant C-glycosyltransferase UrdGT2 was still ambigu-ous.[8,11]

Here we describe our attempts to further investigateUrdGT2, to identify its acceptor substrate, and to possiblygenerate C-glycosidic landomycin derivatives through heter-ologous expression of the corresponding gene urdGT2. Ourwork has showed that the C-glycosylation in the urdamycinbiosynthesis occurs prior to the two aglycon oxygenationsthrough oxygenases UrdE and UrdM, and that the earlyintermediate UWM6 serves as the acceptor substrate forUrdGT2.

Regarding the oxygenases of the landomycin biosynthesis,it was found that oxygenase Lan/LndZ5 is responsible for the11-hydroxylation,[5] and Lan/LndM2 for the 6-hydroxyl-ation,[12] while Lan/LndE most likely catalyzes the 12-oxy-genation (quinone formation), which was assumed to be thefirst oxygenation step in the landomycin biosynthesis. Alloxygenations steps except the 11-hydroxylation occur beforeLan/LndGT2 attaches the first sugar moiety. Thus we firstwanted to confirm these conclusions regarding the oxygen-ation sequence in landomycin biosynthesis by inactivation oflndE, leading to the early block mutant S. globisporus DlndE.This mutant of the landomycin E producer accumulatesprejadomycin (2,3-dehydro-UWM6; 6), an early intermediate

of various angucyclines and angucycline-derived compoundsthat was first discovered in the context of the jadomycinbiosynthesis; more recently it was was also found in blockedmutants of the gilvocarcin V and the oviedomycin producers.The S. globisporus DlndE was obtained by directed disruptionof the lndE gene within the chromosome of S. globisporusSmu622 by insertion of the hygromycin resistance cassette.The accumulation of 6 clearly supports the conclusion thatLndE is the first acting oxygenase of the landomycin pathway.Moreover, the mutant S. globisporus DlndE appeared to bean ideal host for a heterologous expression of urdGT2, thegene encoding C-GT in urdamycin biosynthesis, since itprovided prejadomycin (6), a compound almost identical toUWM6 (10), which was recently discussed as a possibleacceptor substrate of UrdGT2,[19] as well as NDP-activated d-

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olivose, its sugar donor substrate. Moreover, the constructionof an S. globisporus DlndE (urdGT2) mutant not onlypromised to provide information about the acceptor substrateof UrdGT2 but also would allow us to further test thepromiscuity of the other lnd/lan glycosyltransferases, two ofwhich (LndGT1 and LndGT4) were present in the constructalong with their required NDP-activated sugar donor sub-strates. Finding GTs that can elongate a sugar moiety into di-,tri-, or longer saccharide chains regardless of where and howthe first sugar is attached, is important for future saccharideconstructs by combinatorial biosynthesis. While one suchexperiment was already successfully completed with LanGT1,which is closely related to LndGT1,[19] LndGT4/LanGT4 werenever tested on unnatural acceptor substrates.

The S. globisporus DlndE (urdGT2) recombinant strainwas constructed by introduction of plasmid pUWLurdGT2harboring the urdGT2 gene, which was controlled by thePermE promoter through intergeneric conjugation into theDlndE mutant. The presence of the plasmid was proven byphysical isolation, transfer into E. coli XL1-blue MRF,restriction enzyme mapping, Southern blotting with urdGT2amplified from the urdamycin producer S. fradiae TF2717,and sequencing of the PCR product obtained from theplasmid with suitable primers for urdGT2. The resultingconstruct S. globisporus DlndE (urdGT2) was cultivated in4 L of a soya-glucose medium, and the accumulated naturalproducts were isolated. Three novel compounds could beisolated and their structures were elucidated using NMRspectroscopy and mass spectrometry. The mass and UVspectra revealed immediately the chromophore of the poly-ketide moiety to be prejadomycin.[25–27] Typical for thischromophore are the absorption maxima at l= 405 nm and266 nm. Furthermore, the mass spectra data reveal also thepresence of three, two, and one deoxysugar moiety, respec-tively, because of characteristic fragmentations ([M�115]=M�rhodinose for the monosaccharide, [M�115�130]=M�rhodinose�olivose for the disaccharide, and[M�115�130�130]=M�rhodinose�olivose�olivose for thetrisaccharide). Comparison of the NMR data with those ofprejadomycin and the known landomycins[5] indicated thestructures to be 9-C-b-d-olivosylprejadomycin (7, 3 mg), 9-C-b-d-olivosyl-1,4-b-d-olivosylprejadomycin (8, 9 mg), and 9-C-a-l-rhodinosyl-1,3-b-d-olivosyl-1,4-b-d-olivosylprejadomycin(9, 4.5 mg). The conformation and connectivity of the sugarunits (to each other and to the polyketide core) wasconfirmed from 2D NMR experiments (NOESY andCIGAR-HMBC).[28,29] For instance, the anomeric 1A-H ofthe C-glycosidically linked d-olivose unit shows NOE cou-plings with 10-H, 2A-He, 3A-H, and 5A-H; and 3JC,H couplingscan be observed in the CIGAR-HMBC between 1A-H andC8, C10 and C3A along with a weaker 2JC,H coupling with C9(see the Supporting Information). This clearly determines thissugar to be in the 4C1 conformation typical for d-sugars,attached directly at C9, and, based on the large couplingconstant between 1A-H and 2A-Ha of 3JH,H= 10 Hz, contain-ing a b-glycosidic linkage.

In summary, the heterologous expression of gene urdGT2into the lndE-minus mutant of the landomycin E producerS. globisporus 1912 yielded three novel prejadomycin ana-

logues that differ in their C-glycosidically bound moietiesattached at C9. The sugar residue and oligosaccharidemoieties are the same as those previously found in lando-mycins H (4), D (3), and E (1), but they are attached C-glycosidically at C9 instead of O-glycosidically at C8-O, aposition shift that was expected from using UrdGT2. Theglycosyl transfer step occurred on an early angucyclinoneintermediate, prejadomycin (6), also called 2,3-dehydro-UWM6, which was found to be an intermediate of severalpathways. The heterologously expressed C-glycosyl trans-ferase UrdGT2 from the urdamycin biosynthetic pathway,but not its natural competitor, the O-GT LndGT2, which wasalso present, was able to glycosylate compound 6, therebyredirecting the attachment of the landomycin sugar chaintowards the C9-position. The GTs responsible for theelongation to the landomycin E trisaccharide, LndGT4 andLndGT1, could attach their sugars although the aglycon wasstructurally quite different and the first sugar unit was C-attached and at different position. Our results stronglysuggest that UrdGT2 naturally acts on UWM6 (10,Scheme 1) as its acceptor substrate, which differs fromprejadomycin (6) only by its 3-OH group. Thus the ambiguityis resolved regarding the previously not clearly identifiedacceptor substrate for UrdGT2, and also the C-GT step inurdamycin biosynthesis is indicated to most likely occur priorto the two aglycon oxygenation steps catalyzed by UrdE andUrdM (see Scheme 1).[8,19]

To find out whether prejadomycin (6) is an earlyintermediate of the landomycin pathway or an early inter-mediate of a shunt pathway branching from the landomycinpathway (Scheme 1), we performed a crossfeeding experi-ment, in which prejadomycin (6, obtained from Streptomycesglobisporus DlndE) was fed to an early block mutant of thelandomycin biosynthetic pathway (the lndF mutant F133).[12]

In this mutant the PKS-associated fourth ring cyclase genelndF was inactivated, and consequently it cannot produce anyuseful polyketides. But the mutant expresses all downstreamenzymes necessary for the completion of landomycin Ebiosynthesis when it is fed with an intermediate containinga completely cyclized polyketide intermediate. Strain S. glo-bisporus F133 was cultivated for 24 h, and prejadomycin (6)was fed in a single portion (2 mg). HPLC-MS monitoring(every 12 h) showed that significant amounts of landomycin Ewere produced. This clearly proves that prejadomycin (6) isalso an intermediate of the landomycin pathway but cannotbe glycosylated by LndGT2, since this enzyme acts strictly ona later intermediate, in which two of three oxygenation eventshave already occurred. Thus, the urdamycin and landomycinpathways differ significantly with regard to their first glyco-sylation step. For the landomycin biosynthesis, earlier evi-dence was found that the first glycosylation step, the O-glycosylation at 8-O with d-olivose catalyzed by LndGT2,occurred prior to the 11-hydroxylation, catalyzed by LndZ4/Z5, but after the oxygen atoms had been introduced into boththe 12- and 6-positions by LndE and LndM2, respectively.[5] Inparticular, it was recently shown that the non-glycosylatedangucyclinones tetrangomycin (11) and rabelomycin (12) canbe further converted into landomycin E (1) by the lndFmutant F133, and thus 11 and 12 are biosynthetic intermedi-

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ates.[12] The results described here confirm this description ofa—compared to the urdamycin pathway—later first glyco-sylation step. The fact that none of the new prejadomycin C-glycosides including monoglycoside 7 could be furthermodified by the oxygenases that exist in S. globisporusDlndE (urdGT2), for example, by LndM2 and LndZ4/Z5,shows that these oxygenases require non-glycosylated sub-strates. The finding that a compound without 3-OH group,namely 6, serves as an earlier intermediate than twocompounds with 3-OH groups, namely 11 and 12, seemscontradictive. However, in studies with the overexpressedoxygenase JadH, an enzyme responsible for the 12-oxygen-ation in the jadomycin pathway,[25,26] prejadomycin (6) was inpart directly converted into rabelomycin (12), which could beexplained only by a rearrangement initiated by a Michaeladdition of the 4a-OH group at the 3-position.[31] Such arearrangement was also observed by Hutchinson et al. but notexplained.[30] Scheme 1 shows this rearrangement and illus-trates the most probable sequences of the urdamycin andlandomycin pathways. It also includes the “hybrid” pathwayto the new prejadomycin C-glycosides that was initiated bythe heterologously expressed UrdGT2.

Received: August 4, 2006Published online: October 24, 2006

.Keywords: biosynthesis · combinatorial biosynthesis ·glycosides · landomycins · polyketides

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Scheme 1. Biosynthetic pathways for landomycin (black) and urdamycin (blue) and the hybrid pathway to the C-glycosylated prejadomycins 7–9(green). The early urdamycin biosynthetic pathway (blue) proceeds via the hypothetical intermediates 14 and 15.

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