Functional Characterization of tlmK Unveiling Unstable Carbinolamide Intermediates in the Tallysomycin Biosynthetic Pathway
2009; Elsevier BV; Volume: 284; Issue: 13 Linguagem: Inglês
10.1074/jbc.m900640200
ISSN1083-351X
AutoresLiyan Wang, Meifeng Tao, Evelyn Wendt-Pienkoski, Ute Galm, Jane M. Coughlin, Ben Shen,
Tópico(s)Synthetic Organic Chemistry Methods
ResumoTallysomycins (TLMs) belong to the bleomycin family of anticancer antibiotics. TLMs differ from bleomycins primarily by the presence of a 4-amino-4,6-dideoxy-l-talose sugar attached to C-41 as part of a glycosylcarbinolamide. We previously proposed, on the basis of bioinformatics analysis of the tlm biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158, that the tlmK gene is responsible for the attachment of this sugar moiety. We now report that inactivation of tlmK in S. hindustanus abolished TLM A and TLM B production, the resultant ΔtlmK mutant instead accumulated five new metabolites, and introduction of a functional copy of tlmK to the ΔtlmK mutant restored TLM A and TLM B production. Two major metabolites, TLM K-1 and TLM K-2, together with three minor metabolites, TLM K-3, TLM K-4, and TLM K-5, were isolated from the ΔtlmK mutant, and their structures were elucidated. These findings provide experimental evidence supporting the previous functional assignment of tlmK to encode a glycosyltransferase and unveil two carbinolamide pseudoaglycones as key intermediates in the TLM biosynthetic pathway. TlmK stabilizes the carbinolamide intermediates by glycosylating their hemiaminal hydroxyl groups, thereby protecting them from hydrolysis during TLM biosynthesis. In the absence of TlmK, the carbinolamide intermediates fragment to produce an amide TLM K-1 and aldehyde intermediates, which undergo further oxidative fragmentation to afford carboxylic acids TLM K-2, TLM K-3, TLM K-4, and TLM K-5. Tallysomycins (TLMs) belong to the bleomycin family of anticancer antibiotics. TLMs differ from bleomycins primarily by the presence of a 4-amino-4,6-dideoxy-l-talose sugar attached to C-41 as part of a glycosylcarbinolamide. We previously proposed, on the basis of bioinformatics analysis of the tlm biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158, that the tlmK gene is responsible for the attachment of this sugar moiety. We now report that inactivation of tlmK in S. hindustanus abolished TLM A and TLM B production, the resultant ΔtlmK mutant instead accumulated five new metabolites, and introduction of a functional copy of tlmK to the ΔtlmK mutant restored TLM A and TLM B production. Two major metabolites, TLM K-1 and TLM K-2, together with three minor metabolites, TLM K-3, TLM K-4, and TLM K-5, were isolated from the ΔtlmK mutant, and their structures were elucidated. These findings provide experimental evidence supporting the previous functional assignment of tlmK to encode a glycosyltransferase and unveil two carbinolamide pseudoaglycones as key intermediates in the TLM biosynthetic pathway. TlmK stabilizes the carbinolamide intermediates by glycosylating their hemiaminal hydroxyl groups, thereby protecting them from hydrolysis during TLM biosynthesis. In the absence of TlmK, the carbinolamide intermediates fragment to produce an amide TLM K-1 and aldehyde intermediates, which undergo further oxidative fragmentation to afford carboxylic acids TLM K-2, TLM K-3, TLM K-4, and TLM K-5. Tallysomycins (TLMs) 3The abbreviations used are: TLMs, tallysomycins; BLMs, bleomycins; 1H-1H COSY, 1H-1H correlation spectroscopy; ESI, electrospray ionization; HPLC, high performance liquid chromatography; MS, mass spectroscopy; MALDI, matrix-assisted laser desorption ionization; LC, liquid chromatography. belong to the bleomycin (BLM) family of glycopeptide antitumor antibiotics (1Kawaguchi H. Tsukiura H. Tomita K. Konishi M. Saito K. J. Antibiot. (Tokyo). 1977; 30: 779-788Crossref PubMed Scopus (46) Google Scholar, 2Konishi M.S.K. Numata K. Tsuno T. Asama K. J. Antibiot. (Tokyo). 1977; 30: 789-805Crossref PubMed Scopus (69) Google Scholar) (Fig. 1). The BLMs are currently used clinically under the trade name Blenoxane® in combination with a number of other agents for the treatment of Hodgkin lymphoma, carcinomas of the skin, head, and neck, and testicular cancers. Early development of drug resistance and dose-dependent pulmonary toxicity are major limitations of BLMs in chemotherapy (3Chen J. Stubbe J. Nat. Rev. Cancer. 2005; 5: 102-112Crossref PubMed Scopus (484) Google Scholar, 4Hecht S.M. J. Nat. Prod. 2000; 63: 158-168Crossref PubMed Scopus (436) Google Scholar). Structural modifications to this family of natural products are necessary for improvement in efficacy and reduction of toxicity. Although numerous BLM analogs have been synthesized (5Boger D.L. Cai H. Angew. Chem. Int. Ed. 1999; 38: 448-476Crossref PubMed Scopus (0) Google Scholar, 6Leitheiser C.J. Smith K.L. Rishel M.J. Hashimoto S. Konishi K. Thomas C.J. Li C. McCormick M.M. Hecht S.M. J. Am. Chem. Soc. 2003; 125: 8218-8227Crossref PubMed Scopus (47) Google Scholar), total synthesis remains of limited practical value because of the complex scaffold of this entire family of natural products. Recent cloning and characterization of the BLM, TLM, and zorbamycin biosynthetic gene clusters from Streptomyces verticillus ATCC15003 (7Shen B. Du L. Sanchez C. Edwards D.J. Chen M. Murrell J.M. J. Nat. Prod. 2002; 65: 422-431Crossref PubMed Scopus (60) Google Scholar), Streptoalloteichus hindustanus E465-94 (ATCC 31158) (8Tao M. Wang L. Wendt-Pienkowski E. George N.P. Galm U. Zhang G. Coughlin J.M. Shen B. Mol. Biosyst.. 2007; 3: 60-74Crossref PubMed Google Scholar), and Streptomyces flavoviridis ATCC21892 (9Galm U. Wendt-Pienkowski E. Wang L. Geor-ge N. Oh J. Yi F. Tao M. Coughlin J. Shen B. Mol. Biosyst. 2009; 5: 77-90Crossref PubMed Google Scholar) opened the possibility toward the production of novel BLM analogs via genetic metabolic engineering approaches.TLMs are structurally related to BLMs but differ from BLMs in three ways; (i) the presence of two hydroxyl groups within the aminoethylbithiazole moiety, one of which is conjugated to a 4-amino-4,6-dideoxy-l-talose sugar as part of a glycosylcarbinolamide, (ii) the presence of two series of C-terminal amine moieties with (A series) or without (B series) a β-lysine moiety in the subterminal position, and (iii) the absence of a methyl group in the valerate moiety (Fig. 1). One of the B-series TLM analogs, TLM S10b, which has a 1,4-diaminobutane as the C-terminal amine moiety, exhibited antitumor activity similar to that of the BLMs in preclinical studies but failed to yield the desired response in phase II clinical trials due to poor cell penetration (10Nicaise C. Ajani J. Goudeau P. Rozencweig M. Levin B. Krakoff I. Cancer Chemother. Pharmacol. 1990; 26: 221-222Crossref PubMed Scopus (7) Google Scholar, 11Nicaise C. Hong W.K. Dimery W. Usakewicz J. Rozencweig M. Krakoff I. Investig. New Drugs. 1990; 8: 325-328Crossref PubMed Scopus (8) Google Scholar). The results of preclinical and clinical studies indicate that the sugar moieties in the BLM family compounds take part in cellular recognition and drug uptake. Because one of the main differences between the BLMs and TLMs is the presence of a third sugar moiety in the TLMs, we sought to generate the des-talose TLMs to determine whether the talose moiety is responsible for the poor cell penetration of TLM S10b.It is apparent upon comparing and contrasting the genes identified within the tlm and blm clusters that both clusters contain the genes proposed to be responsible for the synthesis and attachment of the l-gulose-3-O-carbamoyl-d-mannose disaccharide. At the downstream part of the tlm gene cluster, however, there is a tlmHJK operon, the homolog of which is absent in the blm cluster. This small operon has been proposed to be involved in the biosynthesis of the 4-amino-4,6-dideoxy-l-talose and its attachment to the TLM nonribosomal peptide skeleton. TlmK, which shows low sequence homology to known glycosyltransferases, was proposed to catalyze the attachment of the talose sugar to the aminoethylbithiazole moiety (8Tao M. Wang L. Wendt-Pienkowski E. George N.P. Galm U. Zhang G. Coughlin J.M. Shen B. Mol. Biosyst.. 2007; 3: 60-74Crossref PubMed Google Scholar). However, the sequence of attachments of the sugar moieties, the l-gulose-3-O-carbamoyl-d-mannose disaccharide and the 4-amino-4,6-dideoxy-l-talose, could not be predicted by bioinformatics analysis alone.The aims of the present study were (i) to provide genetic proof for the role of tlmK in TLM biosynthesis, (ii) to determine the sequence of the sugar attachments, and (iii) to engineer TLM analogs that specifically lack the talose moiety. Here we report that inactivation of tlmK in S. hindustanus abolished TLM A and TLM B production, the resultant ΔtlmK mutant instead accumulated five new metabolites, and introduction of a functional copy of tlmK to the ΔtlmK mutant restored TLM A and TLM B production. Isolation and structural characterization of the five metabolites support the previous functional assignment of tlmK to encode a glycosyltransferase, unveil two carbinolamide pseudoaglycones as key intermediates in the TLM biosynthetic pathway, and suggest the TlmK-catalyzed glycosylation most likely occurred after the attachment of the disaccharide moiety to the TLM aglycone.EXPERIMENTAL PROCEDURESBacterial Strains, Plasmids, and Culture Conditions-S. hindustanus E465-94 ATCC31158 (American Type Culture Collection, Manassas, VA) and derived recombinant strains were routinely cultivated at 30 °C on ISP4 agar medium supplemented with MgCl2 to final concentration of 28 mm or TSB liquid medium. Escherichia coli DH5α and ET12567 (12MacNeil J. Gewain M. Ruby L. Dezeny G. Gibbons H. MacNeil T. Gene (Amst.). 1992; 111: 61-68Crossref PubMed Scopus (589) Google Scholar) were grown in liquid or on solid Luria-Bertani medium at 37 °C (13Sambrook J. Russell W. Molecular Cloning: A Laboratory Manual.3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York2001Google Scholar). E. coli BW25113/pIJ790 and BT340 were cultivated according to the λ RED-mediated PCR-targeting mutagenesis kit (14Gust B. Challis L. Fowler K. Kieser T. Chater F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1541-1546Crossref PubMed Scopus (1191) Google Scholar). Electroporation using S. hindustanus spores was carried out as previously described (8Tao M. Wang L. Wendt-Pienkowski E. George N.P. Galm U. Zhang G. Coughlin J.M. Shen B. Mol. Biosyst.. 2007; 3: 60-74Crossref PubMed Google Scholar).pIJ780 from the λ RED-mediated PCR-targeting mutagenesis kit (14Gust B. Challis L. Fowler K. Kieser T. Chater F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1541-1546Crossref PubMed Scopus (1191) Google Scholar) was modified by replacing the viomycin resistance gene (vph) with the kanamycin-neomycin resistance gene (neo) from Supercos1 (Stratagene, La Jolla, CA) to yield pBS8010. pBS8010 was then used as template to amplify the FRT-neo cassette for replacing tlmK in pBS8008 (8Tao M. Wang L. Wendt-Pienkowski E. George N.P. Galm U. Zhang G. Coughlin J.M. Shen B. Mol. Biosyst.. 2007; 3: 60-74Crossref PubMed Google Scholar) following the λ RED-mediated PCR-targeting mutagenesis protocol. Cosmid pBS8008 carrying 41.6-kilobase S. hindustanus genomic DNA encoding part of the tlm biosynthetic gene cluster was used to construct the gene deletion mutant allele in E. coli by the λ RED-mediated PCR-targeting mutagenesis method. The aac(3)IV-tsr apramycin and thiostrepton resistance gene cassette in its vector backbone was used for selection in both E. coli and S. hindustanus. pBS8004 (8Tao M. Wang L. Wendt-Pienkowski E. George N.P. Galm U. Zhang G. Coughlin J.M. Shen B. Mol. Biosyst.. 2007; 3: 60-74Crossref PubMed Google Scholar) carrying the ΦC31 integration function region and the thiostrepton resistance marker tsr were used to construct the tlmK expression plasmid (supplemental Tables S1 and S2).Construction of the ΔtlmK In-frame Deletion Mutant Strain SB8003-The ΔtlmK in-frame deletion mutant was constructed via a homologous recombination strategy. First, a tlmK allele in pBS1011 was constructed in E. coli by the λ RED-mediated PCR-targeting mutagenesis strategy from pBS8008 using oligonucleotides tlmK-frt1 and tlmK-frt2 (supplemental Table S3). This replaced the tlmK gene in pBS8008 with the FRT-neo cassette amplified from pBS8010 to afford pBS8011. Next, the FRT-neo cassette of pBS8011 was removed by the FLP recombination function provided by E. coli BT340 according to the instructions of the λ RED-mediated PCR-targeting mutagenesis kit (14Gust B. Challis L. Fowler K. Kieser T. Chater F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1541-1546Crossref PubMed Scopus (1191) Google Scholar). In the resulting cosmid pBS8012, the entire tlmK open reading frame was deleted, leaving only the ATG start codon, the TGA stop codon, and an 81-bp stretch of unrelated nucleotides between the ATG and TGA codons. Finally, pBS8012 was passed through E. coli ET12567 to produce demethylated plasmid DNA and subsequently transferred into S. hindustanus wild-type strain by electroporation. Thiostrepton-resistant transformants were selected and underwent one round of nonselective sporulating growth; resulting spores were plated out and screened for thiostrepton-sensitive colonies.The genotype of these thiostrepton-sensitive isolates was determined by PCR using primers tlmK-up and tlmK-down (supplemental Table S3), resulting in identification of five Δtlmk in-frame deletion mutant isolates named SB8003. The genotype of SB8003 was further confirmed by Southern hybridization using the 2238-bp fragment amplified by PCR with primers tlmK-up and tlmK-down (supplemental Table S3) from the S. hindustanus wild-type genomic DNA as a probe. DNA isolations and manipulations were carried out according to standard protocols (13Sambrook J. Russell W. Molecular Cloning: A Laboratory Manual.3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York2001Google Scholar, 15Kieser T. Bibb J. Buttner J. Chater F. Hopwood A. Practical Streptomyces Genetics.2nd Ed. John Innes Foundation, Norwich, UK2000Google Scholar). Southern hybridization using digoxigenin-labeled DNA probes was performed according to manufacturer provided protocols (Roche Diagnostics).Genetic Complementation of the ΔtlmK In-frame Deletion Mutant Strain SB8003-The ErmE* promoter was amplified from pWHM79 by PCR using primers PermEI-f and PermEI-r (supplemental Table S3) and cloned as an EcoRI-BamHI fragment into the same sites of pBS8004 to yield the integrative vector pBS8013. The tlmK gene was amplified from pBS8008 using primers tlmK-NsiI and tlmK-XbaI (Table S3) and cloned as an NsiI-XbaI fragment into the same sites of pBS8013 to afford pBS8014. pBS8014 was introduced into SB8003 by electroporation, and selection with thiostrepton resistance afforded the ΔtlmK-complemented recombinant strain SB8004.Production, Isolation, and Analyses of TLM A, B, K-1, K-2, K-3, K-4, and K-5-The S. hindustanus wild-type strain, the ΔtlmK mutant strain SB8003, and the ΔtlmK-complemented recombinant strain SB8004 were cultured in 50 ml of production medium as described previously (8Tao M. Wang L. Wendt-Pienkowski E. George N.P. Galm U. Zhang G. Coughlin J.M. Shen B. Mol. Biosyst.. 2007; 3: 60-74Crossref PubMed Google Scholar). After centrifugation, the supernatant (∼40 ml) was adjusted to pH 7.0 with 1.0 m HCl and mixed with Amberlite® IRC50 resin (H+-type, 10 ml). After incubation of the resultant slurry at room temperature with gentle agitation for 30 min, the Amberlite® IRC-50 resin was packed into a column, washed with 10 bed-volumes of water, and drained of excess water. TLM A and B from the wild-type ATCC 31158 and the recombinant SB8004 strains and TLM K-1, K-2, K-3, K-4, and K-5 from the ΔtlmK mutant SB8003 strain were eluted with 30 ml of 0.2 m HCl. The Amberlite® IRC50 eluent was neutralized with 1.0 n NaOH and concentrated in vacuo to ∼1 ml. Analytic HPLC was carried out on an Apollo C-18 column (5 μm, 250 × 4.6 mm, Alltech Associates, Inc., Deerfield, IL). The column was equilibrated with 100% solvent A (99.8% H2O, 0.2% acetic acid) and 0% solvent B (99.8% methanol, 0.2% acetic acid) and developed with a linear gradient (0–5 min, linear gradient from 100% A/0% B to 90% A/10% B; 5–30 min, linear gradient from 90%A/10% B to 0% A/100% B; 30–35 min, 0% A/100% B) at a flow rate of 0.7 ml/min and UV detection at 300 nm using a Varian Prostar 330 PDA detector (Varian, Palo Alto, CA). Under these conditions, TLM A and B were eluted with retention time of 11.0 and 12.4 min, respectively, whereas TLM K-1, K-2, K-3, K-4, and K-5 were eluted with retention time of 10.4, 15.2, 15.8, 16.3, and 16.9 min, respectively.For preparative-scale isolation of the TLM K-1, K-2, K-3, K-4, and K-5 from the ΔtlmK mutant SB8003 strain, the fermentation culture (10 liters) was centrifuged at 3000 rpm for 30 min, and the supernatant was collected, adjusted to pH 7.0 with 1.0 m HCl, and loaded onto an Amberlite® XAD-16 column (1.0 liter). After washing the column with three bed volumes of H2O, these intermediates were eluted with 2.0 liters of 80% MeOH. The resulting eluent was concentrated in vacuo to ∼10 ml and then loaded to a CM-Sephadex C-25 (50 × 20 mm) column. The column was washed with 3 bed-volumes of H2O and eluted with 0.1, 0.2, 0.3, 0.5, or 1.0 m NH4OAc sequentially. Fractions containing TLM K-1 were eluted at 0.1 m NH4OAc, fractions containing TLM K-4 and K-5 were eluted at 0.2 m NH4OAc, fractions containing TLM K-2 were eluted at 0.3 m NH4OAc, and fractions containing TLM K-3 were eluted at 0.5 m NH4OAC. Fractions containing each metabolite were re-loaded onto Amberlite® XAD-16 columns (50 × 20 mm). The columns were washed with three bed-volumes of H2O and then eluted with three bed-volumes of 80% MeOH. The MeOH eluents were then concentrated in vacuo to ∼2 ml.Final purification of each metabolite was achieved by semi-preparative HPLC on an Altima C18 column (5 μm, 250 × 10 mm, Alltech Associates, Inc.). HPLC was carried out under the following conditions. Instrument and detector were the same as stated above. The column was equilibrated with 100% solvent A (0.1% formic acid) and 0% solvent B (methanol) and developed with a linear gradient (0–15 min, from 95% A/5% B to 50% A/50% B; 15 to 18 min, from 50% A/50% B to 0% A/100% B) at a flow rate of 3 ml/min and with UV detection at 300 nm. TLM K-1, K-2, K-3, and K-5 were eluted with retention times of 5.0, 7.5, 7.7, and 8.5 min, respectively. Upon final removal of MeOH by concentration in vacuo, the residues were lyophilized to afford the final pure metabolites TLM K-1 (as copper complex, 4.1 mg), TLM K-2 (1.4 mg), TLM K-3 (0.5 mg), and TLM K-5 (0.3 mg), respectively. Final treatment of the TLM K-1 copper complex with 0.5 m EDTA-Na (pH 7.3) solution followed by a HPLC purification step afforded copper-free TLM K-1 (3.2 mg) (8Tao M. Wang L. Wendt-Pienkowski E. George N.P. Galm U. Zhang G. Coughlin J.M. Shen B. Mol. Biosyst.. 2007; 3: 60-74Crossref PubMed Google Scholar, 9Galm U. Wendt-Pienkowski E. Wang L. Geor-ge N. Oh J. Yi F. Tao M. Coughlin J. Shen B. Mol. Biosyst. 2009; 5: 77-90Crossref PubMed Google Scholar).Each of the purified metabolites was subjected to MS, one-dimensional and two-dimensional 1H or 13C NMR spectroscopic analyses, or a combination of thereof for structural determination. LC-MS analysis was carried out on an Agilent 1100 HPLC-MSD SL quadrupole mass spectrometer (Santa Clara, CA), and high resolution matrix-assisted laser desorption ionization (MALDI)-Fourier transform MS analysis was carried out on an IonSpec HiResMALDI Fourier transform mass spectrometer with a 7-tesla superconducting magnet (Lake Forest, CA). 1H and 13C NMR spectra were obtained on a Varian Unity Inova 500 instrument at 500 MHz for 1H and 125 MHz for 13C nuclei, and two-dimensional NMR spectra were performed using standard Varian pulse sequences (Palo Alto, CA).RESULTSCharacterization of tlmK by Gene Inactivation and Mutant Complementation-To investigate its role in TLM biosynthesis, an in-frame deletion of the tlmK gene was generated in the TLM producing S. hindustanus wild-type strain by homologous recombination (Fig. 2A). pBS8012, carrying a ΔtlmK in-frame deletion mutant allele, was made from pBS8008, a cosmid carrying 41.6 kilobases of downstream part of the tlm gene cluster. pBS8012 was then transformed into S. hindustanus wild-type strain by electroporation followed by screening for double crossover homologous recombination events, yielding five isolates of the mutant strain SB8003; the ΔtlmK in-frame deletion genotype in SB8003 was confirmed by Southern hybridization (Fig. 2B). Fermentations of SB8003 showed that (i) TLM A and B production has been abolished and, (ii) instead, five new metabolites were found with retention times of 10.4 (TLM K-1), 15.2 (TLM K-2), 15.8 (TLM K-3), 16.3 (TLM K-4), and 16.9 (TLM K-5) min, which were apparently different from those of TLM A and B (Fig. 2C).FIGURE 2Inactivation of tlmK and genetic complementation to the ΔtlmK in-frame deletion mutant. A, construction of the ΔtlmK mutant strain SB8003 and restriction maps of the S. hindustanus wild-type and SB8003 mutant strains as well as the pBS8012 cosmid carrying the ΔtlmK in-frame deletion mutation upon XcmI digestion. B, Southern analysis of the ΔtlmK mutant strain SB8003 (lanes 1, 2, 3, 4, and 5 for five independent isolates) with the ΔtlmK mutant construct pBS8012 (lane 6) and tlmK wild-type construct pBS8008 (lane 7) as controls upon XcmI digestion and using the 2.2-kilobase (kb) PCR-amplified fragment from the wild-type strain as a probe. C, HPLC analysis of (a) TLM production in S. hindustanus wild type, (b) new metabolite accumulation in the ΔtlmK mutant strain SB8003, and (c) restoration of TLM production in the ΔtlmK-complemented recombinant strain SB8004. ◯, TLM A; □, TLM B; •, TLM K-1; ▪, TLM K-2; ▴, TLM K-3; ♦, TLM K-4; ▾, TLM K-5. mAU, milliabsorbance.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To genetically complement the ΔtlmK mutation in SB8003, pBS8014, an integrative plasmid carrying a functional copy of tlmK, whose expression is under control of the constitutive ErmE* promoter (16Bibb M. White J. Ward J. Janssen J. Mol. Microbiol. 1994; 14: 533-545Crossref PubMed Scopus (162) Google Scholar), was conjugated into SB8003 to yield the complementation strain SB8004 (i.e. SB8003 (pBS8014)). The production of TLM A and B was partially restored in SB8004 with TLM A and B titers of 8–10 mg/liter, which is ∼50% that for the S. hindustanus wild-type strain (Fig. 2C).Isolation and Structural Characterization of the New Metabolites Accumulated in the ΔtlmK Mutant Strain SB8003-Deletion of tlmK, thereby inactivating the TlmK glycosyltransferase in TLM biosynthesis, might lead to the production of a carbinolamide intermediate lacking the talose moiety at C-41 (Fig. 1). Such intermediates may readily undergo fragmentation to afford an amide and aldehyde (see Fig. 4). As anticipated, one of the five new metabolites accumulated by SB8003 showed a [M+Cu-H]+ ion at m/z 1122.3 upon LC-ESI-MS analysis, corresponding to the calculated molecular weight of the amide species TLM K-1. The other metabolites showed [M+H]+ ions at m/z 542.2, 468.0, 370.1, and 340.1 upon LC-ESI-MS analysis, but none of them matched the molecular weight of the proposed aldehyde species. Instead, the ions at m/z 542.2 (TLM K-2) and 370.1 (TLM K-4) matched those of carboxylic acids oxidized from the predicted aldehydes, whereas the ions at m/z 468.0 (TLM K-3) and 340.1 (TLM K-5) matched the further decomposed products of TLM K-2 and TLM K-4, respectively. The estimated yields of TLM K-1, K-2, K-3, K-4, and K-5 are ∼5.0, 3.0, 1.0, 0.5, and 0.3 mg/liter, respectively.FIGURE 4Biosynthesis of TLMs featuring the carbinolamide pseudoaglycones as key intermediates, which in the absence of the TlmK glycosyltransferase, undergo rapid degradation into TLM K-1, K-2, K-3, K-4, and K-5.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To establish the structures, these metabolites were isolated from large scale fermentation (10 liter). TLM K-1 was isolated as a blue powder, which is characteristic for members of the BLM and TLM family as a copper complex. To remove copper for NMR analysis, the complex was treated with EDTA to afford copper-free TLM K-1 as a pale white powder. The high resolution MALDI-Fourier transform mass spectrometry analysis of copper-free TLM K-1 yielded a[M+H]+ ion at m/z 1061.4496, which corresponded to the molecular formula C40H64N14O20 + H+ (calculated 1061.4499). The 1H and 13C NMR data of copper-free TLM K-1 were almost identical to those of the left portion of TLM A, including the pyrimidoblamic acid, β-hydroxyhistidine, 4-amino-3-hydroxy pentanoic acid, threonine, and 2-O-(3-O-carbamoyl-α-d-mannosyl)-l-gulose moieties but lacked the signals corresponding to the right portion of TLM A, including the 2′-(2-amino-1,2-dihydroxyethyl)-2,4′-bithiazole-4-carboxylic acid, terminal amines (for both A- and B-series of TLMs), and the 4-amino-4,6-dideoxy-l-talose moiety (17Greenaway F.T. Dabrowiak J.C. Grulich R. Crooke S.T. Org. Magnet. Res. 1980; 13: 270-273Crossref Scopus (7) Google Scholar, 18Calafat A. Won H. Marzilli L. J. Am. Chem. Soc. 1997; 119: 3656-3664Crossref Scopus (41) Google Scholar) (Figs. 1 and 4). The structure of TLM K-1 was finally unambiguously determined by careful analysis of 1H, 13C, and two-dimensional NMR measurements as the degraded product, containing the “left” structure units mentioned above, of the proposed carbinolamide intermediates in the TLM biosynthetic pathway (Fig. 3 and Table 1).FIGURE 3Key 1H-1H COSY and heteronuclear multiple bond correlation (HMBC) correlations of TLM K-1, K-2, and K-5.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 113C NMR and 1H NMR data of TLM K-1 [D2O, 3-trimethylsilyl[2,2,3,3-2H4]propionate, δ (ppm) (J = HZ)]No.aH and C numbering is based on TLM A.δCδH1173.8255.24.02 mbOverlapped signal.349.82.97 dd (13.5, 7.0), 3.01 dd (13.5, 5.5)4179.0543.02.66 dd (15.0, 8.5), 2.71 dd (15.0, 3.0)662.84.00 mbOverlapped signal.7161.18167.39114.310156.21113.71.93 s12170.81359.05.11 d (7.5)1475.35.39 d (7.5)15101.65.28 d (4.0)1670.53.98 mbOverlapped signal.1771.93.88 mbOverlapped signal.1870.34.16 mbOverlapped signal.1972.94.06 mbOverlapped signal.20100.95.03 d (2.0)2171.24.13 mbOverlapped signal.2277.34.80 mbOverlapped signal.2367.63.85 mbOverlapped signal.2476.33.84 mbOverlapped signal.2563.83.82 mbOverlapped signal., 3.96 mbOverlapped signal.26161.127134.228121.67.54 s29138.48.53 s30171.53152.73.90 mbOverlapped signal.3217.11.18 d (6.5)3373.64.1 mbOverlapped signal.3442.62.45 dd (14.5, 9.5), 2.50 dd (14.5, 3.0)36177.23761.64.31 d (3.5)3869.94.26 m3921.71.19 d (6.5)40177.85063.33.39 dd (11.5, 7.5), 3.54 dd (11.5, 4.5)a H and C numbering is based on TLM A.b Overlapped signal. Open table in a new tab TLM K-2, K-3, and K-5 were all purified as pale white powders. Although upon ESI-MS analysis TLM K-2 showed a [M+H]+ ion at m/z 542.2, high resolution MALDI Fourier transform MS analysis of TLM K-2 afforded a decarboxylation fragment [M-COOH+H]+ ion at m/z 498.2355, which agrees well with the molecular formula of C21H36N7O3S2 + H+ (calculated 498.2394). The 1H and 13C NMR data of TLM K-2 are almost identical to those of the “right” portion of TLM A (17Greenaway F.T. Dabrowiak J.C. Grulich R. Crooke S.T. Org. Magnet. Res. 1980; 13: 270-273Crossref Scopus (7) Google Scholar, 18Calafat A. Won H. Marzilli L. J. Am. Chem. Soc. 1997; 119: 3656-3664Crossref Scopus (41) Google Scholar) (Fig. 3 and Table 2). However, an oxygen-bearing methine signal (δC 83.3) for C-41 observed in the 13C NMR spectrum of TLM A was absent in that of TLM K-2, and instead, a new carbonyl signal at δC 178.0 was observed. Combined with the decarboxylation fragment detected in the MALDI-MS spectrum of TLM K-2, this carbonyl moiety was considered as a part of a newly formed carboxylic acid group. The correlation between H-42 (δH 5.38 s) and the carbonyl carbon indicated that this carboxylic acid group is located at C-41 (Fig. 3). On the basis of these data the complete structure of TLM K-2 was established. The structure of TLM K-2 indicates that the nascent aldehyde species (right portion), generated upon hydrolysis of the proposed carbinolamide intermediate, is further oxidized spontaneously to form the corresponding carboxylic acid TLM K-2 (Fig. 4).TABLE 213C NMR and 1H NMR data of 2, 3, and 5 [D2O, 3-trimethylsilyl[2,2,3,3-2H4]propionate, δ(ppm) (J = HZ)]No.cH and C numbering is based on TLM A and TLM B.TLM K-2aAssignment confirmed by 1H-1H COSY, total correlation spectroscopy, heteronuclear single quantum coherence, and heteronuclear multiple bond correlation spectra obtained at 500 MHz and 125 MHz.TLM K-3aAssignment confirmed by 1H-1H COSY, total correlation spectroscopy, heteronuclear single quantum coherence, and heteronuclear multiple bond correlation spectra obtained at 500 MHz and 125 MHz.TLM K-5bAssignment confirmed by 1H NMR and 1H-1H COSY obtained at 500 MHz. δHδCδHδCδH41178.0-dNot assigned.4275.25.38 s-43175.9-44149.98.18 s-8.24 s8.21 s45165.7-46165.7-47151.68.21 s-8.32 s8.30 s48166.0-49172.1-5741.473.47 m41.473.49 m3.45 t (6.0)5827.31.75 meOverlapped signal.27.31.75 meOverlapped signal.2.08 m5932.01.77 meOverlapped signal.32.01.77 me3.20 t (6.5)6051.33.66 m51.33.68 m1.82 meOverlapped signal.6139.42.63 m39.42.67 m3.17 m62174.7-1.82 meOverlapped signal
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