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• W2024459080 abstract "We have previously demonstrated that the biosynthesis of the C7-cyclitol, called valienol (or valienamine), of the α-glucosidase inhibitor acarbose starts from the cyclization of sedo-heptulose 7-phosphate to 2-epi-5-epi-valiolone (Stratmann, A., Mahmud, T., Lee, S., Distler, J., Floss, H. G., and Piepersberg, W. (1999)J. Biol. Chem. 274, 10889–10896). Synthesis of the intermediate 2-epi-5-epi-valiolone is catalyzed by the cyclase AcbC encoded in the biosynthetic (acb) gene cluster of Actinoplanes sp. SE50/110. The acbCgene lies in a possible transcription unit, acbKLMNOC, cluster encompassing putative biosynthetic genes for cyclitol conversion. All genes were heterologously expressed in strains ofStreptomyces lividans 66 strains 1326, TK23, and TK64. The AcbK protein was identified as the acarbose 7-kinase, which had been described earlier (Drepper, A., and Pape, H. (1996) J. Antibiot. (Tokyo) 49, 664–668). The multistep conversion of 2-epi-5-epi-valiolone to the final cyclitol moiety was studied by testing enzymatic mechanisms such as dehydration, reduction, epimerization, and phosphorylation. Thus, a phosphotransferase activity was identified modifying 2-epi-5-epi-valiolone by ATP-dependent phosphorylation. This activity could be attributed to the AcbM protein by verifying this activity inS. lividans strain TK64/pCW4123M, expressing His-tagged AcbM. The His-tagged AcbM protein was purified and subsequently characterized as a 2-epi-5-epi-valiolone 7-kinase, presumably catalyzing the first enzyme reaction in the biosynthetic route, leading to an activated form of the intermediate 1-epi-valienol. The AcbK protein could not catalyze the same reaction nor convert any of the other C7-cyclitol monomers tested. The 2-epi-5-epi-valiolone 7-phosphate was further converted by the AcbO protein to another isomeric and phosphorylated intermediate, which was likely to be the 2-epimer 5-epi-valiolone 7-phosphate. The products of both enzyme reactions were characterized by mass spectrometric methods. The product of the AcbM-catalyzed reaction, 2-epi-5-epi-valiolone 7-phosphate, was purified on a preparative scale and identified by NMR spectroscopy. A biosynthetic pathway for the pseudodisaccharidic acarviosyl moiety of acarbose is proposed on the basis of these data. We have previously demonstrated that the biosynthesis of the C7-cyclitol, called valienol (or valienamine), of the α-glucosidase inhibitor acarbose starts from the cyclization of sedo-heptulose 7-phosphate to 2-epi-5-epi-valiolone (Stratmann, A., Mahmud, T., Lee, S., Distler, J., Floss, H. G., and Piepersberg, W. (1999)J. Biol. Chem. 274, 10889–10896). Synthesis of the intermediate 2-epi-5-epi-valiolone is catalyzed by the cyclase AcbC encoded in the biosynthetic (acb) gene cluster of Actinoplanes sp. SE50/110. The acbCgene lies in a possible transcription unit, acbKLMNOC, cluster encompassing putative biosynthetic genes for cyclitol conversion. All genes were heterologously expressed in strains ofStreptomyces lividans 66 strains 1326, TK23, and TK64. The AcbK protein was identified as the acarbose 7-kinase, which had been described earlier (Drepper, A., and Pape, H. (1996) J. Antibiot. (Tokyo) 49, 664–668). The multistep conversion of 2-epi-5-epi-valiolone to the final cyclitol moiety was studied by testing enzymatic mechanisms such as dehydration, reduction, epimerization, and phosphorylation. Thus, a phosphotransferase activity was identified modifying 2-epi-5-epi-valiolone by ATP-dependent phosphorylation. This activity could be attributed to the AcbM protein by verifying this activity inS. lividans strain TK64/pCW4123M, expressing His-tagged AcbM. The His-tagged AcbM protein was purified and subsequently characterized as a 2-epi-5-epi-valiolone 7-kinase, presumably catalyzing the first enzyme reaction in the biosynthetic route, leading to an activated form of the intermediate 1-epi-valienol. The AcbK protein could not catalyze the same reaction nor convert any of the other C7-cyclitol monomers tested. The 2-epi-5-epi-valiolone 7-phosphate was further converted by the AcbO protein to another isomeric and phosphorylated intermediate, which was likely to be the 2-epimer 5-epi-valiolone 7-phosphate. The products of both enzyme reactions were characterized by mass spectrometric methods. The product of the AcbM-catalyzed reaction, 2-epi-5-epi-valiolone 7-phosphate, was purified on a preparative scale and identified by NMR spectroscopy. A biosynthetic pathway for the pseudodisaccharidic acarviosyl moiety of acarbose is proposed on the basis of these data. The α-glucosidase inhibitor acarbose (part of the amylostatin complex) (Fig. 1), produced by strains of the genera Actinoplanes and Streptomyces, is a member of an unusual group of bacterial (mainly actinomycete) secondary metabolites, all of which inhibit various α-glucosidases, especially in the intestine (1Truscheit E. Frommer W. Junge B. Müller L. Schmidt D.D. Wingeder W. Angew. Chem. Int. Ed. Engl. 1981; 20: 744-761Crossref Scopus (567) Google Scholar, 2Müller L. Demain A.L. Somkuti G.A. Hunter-Creva J.C. Rossmoore H.W. Novel Microbial Products for Medicine and Agriculture. Elsevier Science Publishers B.V., Amsterdam1989: 109-116Google Scholar). Acarbose is produced industrially using developed strains of Actinoplanes sp. SE50/110. It is predominantly used in the treatment of diabetes patients, enabling them to better utilize starch- or sucrose-containing diets by slowing down the intestinal release of α-d-glucose. The pseudotetrasaccharide acarbose consists of an unsaturated cyclitol (valienol), a 4-amino-4,6-dideoxyglucose, and maltose. The valienol and 4-amino-4,6-dideoxyglucose are linked via an amino bridge mimicking anN-glycosidic bond. This acarviosyl moiety is primarily responsible for the inhibitory effect on α-glucosidases. Biosynthetically, these compounds resemble aminoglycoside antibiotics (3Piepersberg W. Distler J. Kleinkauf H. von Döhren H. Biotechnology: Products of Secondary Metabolism. 2nd Ed. 7. VCH Verlagsgesellschaft mbH, Weinheim, Germany1997: 397-488Google Scholar, 4Piepersberg W. Strohl W.R. Biotechnology of Antibiotics. 2nd Ed. Marcel Dekker, Inc., New York1997: 81-163Google Scholar). Dependent on the carbon sources in the fermentation medium, Actinoplanes sp. SE50/110 produces also higher homologs of acarbose, which differ in the numbers of glucose residues that are linked to the reducing and nonreducing end of the acarviosyl moiety (Fig. 1). The C7-aminocyclitol units are considered to be similar to other common structural motifs observed in bacterial secondary metabolites (4Piepersberg W. Strohl W.R. Biotechnology of Antibiotics. 2nd Ed. Marcel Dekker, Inc., New York1997: 81-163Google Scholar). The transition from primary to secondary metabolism in the cyclitol pathway in Actinoplanes sp. SE50/110 is catalyzed by the AcbC protein. The acbC gene was expressed heterologously in Streptomyces lividans employing the same reaction conditions as used in in vitro studies on dehydroshikimate synthase (dehydroquinate synthase, AroB) proteins. Its product was shown to be a C7-cyclitol synthase usingsedo-heptulose 7-phosphate as substrate for the production of 2-epi-5-epi-valiolone (5Stratmann A. Mahmud T. Lee S. Distler J. Floss H.G. Piepersberg W. J. Biol. Chem. 1999; 274: 10889-10896Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Until now, no other intermediate for the biosynthesis of acarbose has been identified. The trehalase inhibitor validamycin A (cf. Fig.1 B) is an antifungal antibiotic used as a crop protectant. Validamycins are produced by Streptomyces hygroscopicus ssp.limoneus and consist of two similar C7-cyclitol units, one belonging to the valienol family (valienamine) and the other to a saturated 6-hydroxy derivative thereof (called validamine). In the biosynthetic pathway for validamycin, 2-epi-5-epi-valiolone has also been identified as the first precursor for these two cyclitol units. In this pathway, the feeding of various other potential precursors had led to the identification of some intermediates, including 5-epi-valiolone, valienone, and valienamine (cf.Fig. 1) (6Dong H. Mahmud T. Tornus I. Lee S. Floss H.G. J. Am. Chem. Soc. 2001; 123: 2733-2742Crossref PubMed Scopus (55) Google Scholar, 7Mahmud T., Xu, J. Choi Y.U. J. Org. Chem. 2001; 66: 5066-5073Crossref PubMed Scopus (20) Google Scholar). In contrast, similar feeding experiments revealed 2-epi-5-epi-valiolone to be the only precursor that was incorporated into acarbose (8Mahmud T. Tornus I. Engelkrout E. Wolf E., Uy, C. Floss H.G. Lee S. J. Am. Chem. Soc. 1999; 121: 6973-6983Crossref Scopus (51) Google Scholar). Therefore, fundamental differences in the two pathways leading to the very similar end products are likely to exist. In this study, we show that, during the biosynthesis of acarbose inActinoplanes sp. SE50/110, the cyclitol precursor 2-epi-5-epi-valiolone is phosphorylated, forming the intermediate 2-epi-5-epi-valiolone 7-phosphate, by the enzyme AcbM as a first step in its conversion to the valienol moiety. Beyond this, we found that AcbO catalyzed the next conversion step, leading to an isomeric phosphorylated substance with the same molecular mass, most likely the epimerization product of 2-epi-5-epi-valiolone 7-phosphate to 5-epi-valiolone 7-phosphate. These findings, together with the genetic record from the acb gene cluster, provided evidence for the postulate of a new biosynthetic pathway for the acarviosyl moiety of acarbose, resembling those for activation (by phosphorylation and subsequent nucleotidylation) and modification of hexoses to be incorporated into oligo- or polysaccharides by glycosyl transfer (3Piepersberg W. Distler J. Kleinkauf H. von Döhren H. Biotechnology: Products of Secondary Metabolism. 2nd Ed. 7. VCH Verlagsgesellschaft mbH, Weinheim, Germany1997: 397-488Google Scholar, 4Piepersberg W. Strohl W.R. Biotechnology of Antibiotics. 2nd Ed. Marcel Dekker, Inc., New York1997: 81-163Google Scholar, 9Liu H.W. Thorson J.S. Annu. Rev. Microbiol. 1994; 48: 223-256Crossref PubMed Scopus (238) Google Scholar). The fact of 7-O-phosphorylation in addition points to the need of an inactivating protection group already in the cyclitol intermediates and the oligosaccharidic end product(s) inside the producing cell. This requirement is underlined by the existence of a second 7-phosphotransferase gene, acbK, which is localized in the same transcription unit together with theacbM and other putative cyclitol biosynthetic genes and encodes a cytoplasmic acarbose 7-kinase. AcbK introduces a phosphate group into the same position of the cyclitol moiety of the oligosaccharidic end product, but does not use monomeric cyclitol precursors such as 2-epi-5-epi-valiolone as substrates. The bacterial strains and plasmids used in this study are listed in Table I. The following strains ofS. lividans 66 were used as the hosts in expression experiments for the heterologous production of Acb proteins: strain TK64 for AcbL, AcbM, and AcbO; strain TK23 for AcbK and AcbN; and strain 1326 for AcbC. The recombinant strains were routinely cultured at 28 °C on soya fluor-mannitol-agar (SMA) agar plates (10Distler J. Klier K. Piendl W. Werbitzki O. Böck A. Kresze G. Piepersberg W. FEMS Microbiol. Lett. 1985; 30: 145-150Crossref Scopus (26) Google Scholar), yeast extract-malt extract medium (YEME) medium with 10.3 or 34% sucrose (11Hopwood D.A. Bibb M.J. Chater K.F. Kieser T. Bruton C.J. Kieser H.M. Lydiate D.J. Smith C.P. Ward J.M. Schrempf H. Genetic Manipulation of Streptomyces: A Laboratory Manual. John Innes Institute, Norwich, UK1985Google Scholar), or protoplast regeneration medium (SPMR) plates (12Babcock M.J. Kendrick K.E. J. Bacteriol. 1988; 170: 2802-2808Crossref PubMed Google Scholar); Actinoplanes sp. SE50/110 was cultured in MD50 medium (2Müller L. Demain A.L. Somkuti G.A. Hunter-Creva J.C. Rossmoore H.W. Novel Microbial Products for Medicine and Agriculture. Elsevier Science Publishers B.V., Amsterdam1989: 109-116Google Scholar). To maintain plasmids pIJ4123 and pIJ6021 and their recombinant derivatives, media were supplemented with kanamycin (50 mg/liter). The thiostrepton-inducible expression of the cloned acbC, acbL, acbM, andacbO genes in S. lividans TK64 was carried out according to Takano et al. (13Takano E. White J. Thompson C.J. Bibb M.J. Gene (Amst.). 1995; 166: 133-137Crossref PubMed Scopus (128) Google Scholar), with the exception that thiostrepton was used at a concentration of 10 μg/ml, and the incubation time after induction was prolonged to 24 h. Recombinant Escherichia coli strains were grown at 37 °C in LB broth or on LB agar plates (14Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar) supplemented with ampicillin (100 mg/liter).Table IBacterial strains and plasmidsStrain/plasmidProperties/productSource/Ref.Bacterial strains Actinoplanes sp. SE50/110AcarboseATCC 31044 S. lividans 66 1326Actinorhodin, prodigiosin11Hopwood D.A. Bibb M.J. Chater K.F. Kieser T. Bruton C.J. Kieser H.M. Lydiate D.J. Smith C.P. Ward J.M. Schrempf H. Genetic Manipulation of Streptomyces: A Laboratory Manual. John Innes Institute, Norwich, UK1985Google Scholar S. lividansTK23Actinorhodin, prodigiosinspc-111Hopwood D.A. Bibb M.J. Chater K.F. Kieser T. Bruton C.J. Kieser H.M. Lydiate D.J. Smith C.P. Ward J.M. Schrempf H. Genetic Manipulation of Streptomyces: A Laboratory Manual. John Innes Institute, Norwich, UK1985Google Scholar S. lividansTK64Actinorhodin, prodigiosin spc-2, pro-2, str-611Hopwood D.A. Bibb M.J. Chater K.F. Kieser T. Bruton C.J. Kieser H.M. Lydiate D.J. Smith C.P. Ward J.M. Schrempf H. Genetic Manipulation of Streptomyces: A Laboratory Manual. John Innes Institute, Norwich, UK1985Google Scholar E. coliBL21(DE3)/pLysST7 RNA polymerase,cat21Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6005) Google Scholar E. coliDH5αF ϕ80d, lacZΔM15, endA1, recA1, hsdR17 (rk−mk+), supE44, thi-1, · , gyrA96, relA1, D(lacZYA-argF)U16922Hanahan D. J. Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8190) Google Scholar E. coli JM109 F′ traD36, lacZΔM15,proA + B + lac1 · λ · recA1, hsdR17 (rk− mk+), supE44, thi-1, gyrA96, relA1, Δ(lac −)22Hanahan D. J. Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8190) Google ScholarPlasmids pBluescript II SK bla, lacZ-α, f1 ori24Short J. Fernandez J. Sorge J. Huse W. Nucleic Acids Res. 1988; 16: 7583-7600Crossref PubMed Scopus (1080) Google Scholar pET11aP bla, lacZ-α, T7 promoter25Wehmeier U.F. FEMS Microbiol. Lett. 2001; 197: 53-58Crossref PubMed Google Scholar pET16bP bla, lacZ-α, T7 promoter, His-tagged fusion peptide25Wehmeier U.F. FEMS Microbiol. Lett. 2001; 197: 53-58Crossref PubMed Google Scholar pJOE2702 bla, rrnB, rha-p26Volff J.N. Eichenseer C. Viell P. Piendl W. Altenbuchner J. Mol. Microbiol. 1996; 21: 1037-1047Crossref PubMed Scopus (69) Google Scholar pIJ4123 kan, tsr, tipAp13Takano E. White J. Thompson C.J. Bibb M.J. Gene (Amst.). 1995; 166: 133-137Crossref PubMed Scopus (128) Google Scholar pIJ6021 kan, tsr, tipAp13Takano E. White J. Thompson C.J. Bibb M.J. Gene (Amst.). 1995; 166: 133-137Crossref PubMed Scopus (128) Google Scholar pUC18 bla, lacZ-α23Yanisch-Perron C. Vieira J. Messing J. Gene (Amst.). 1985; 33: 103-119Crossref PubMed Scopus (11465) Google Scholar pUWL201RBSA bla, lacZ-α, tsr, ermEp27Doumith M. Weingarten P. Wehmeier U.F. Salah-Bey K. Benhamou B. Capdevila C. Michel J.-M. Piepersberg W. Raynal M.-C. Mol. Gen. Genet. 2000; 264: 277-285Crossref Scopus (90) Google Scholar pPWW49 bla, lacZ-α, tsr, ermEp, eryBIV27Doumith M. Weingarten P. Wehmeier U.F. Salah-Bey K. Benhamou B. Capdevila C. Michel J.-M. Piepersberg W. Raynal M.-C. Mol. Gen. Genet. 2000; 264: 277-285Crossref Scopus (90) Google Scholar pPWW50 bla, lacZ-α, tsr, ermEp, eryBIV27Doumith M. Weingarten P. Wehmeier U.F. Salah-Bey K. Benhamou B. Capdevila C. Michel J.-M. Piepersberg W. Raynal M.-C. Mol. Gen. Genet. 2000; 264: 277-285Crossref Scopus (90) Google Scholar pAS8/7 acbC in pIJ60215Stratmann A. Mahmud T. Lee S. Distler J. Floss H.G. Piepersberg W. J. Biol. Chem. 1999; 274: 10889-10896Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar pCWL161.42-kb NdeI/SstI fragment from pMJL1 in pET16bP (NdeI/SstI)This work pCWN160.86-kb NdeI/BamHI fragment from pMJN1 in pET16bP (NdeI/BamHI)This work pCWO160.87-kb NdeI/BglII fragment from pMJO1 in pET16bP (NdeI/BamHI)This work pCWM161.08-kb NdeI/KpnI fragment from pMJM1 in pET16bP (NdeI/KpnI)This work pCWK111.0-kb NdeI/BglII acbKfragment in pET11aP (NdeI/BamHI)This work pCWK161.0-kb NdeI/BglII acbKfragment in pET16bP (NdeI/BamHI)This work pCW201L1.1-kb NdeI/HindIIIacbL fragment in pUWL201RBSA (NdeI/HindIII)This work pCW201M1.08-kb NdeI/HindIIIacbM fragment in pUWL201RBSA (NdeI/HindIII)This work pCW201O0.82-kb NdeI/HindIIIacbO fragment in pUWL201RBSA (NdeI/HindIII)This work pCW2072K1.0-kb NdeI/BglIIacbK fragment in pJOE2702 (NdeI/BamHI)This work pCW201KM61.98-kb NdeI/HindIIIacbKM fragment in pUWL201RBSA (NdeI/HindIII)This work pCW4123L1.42-kb NdeI/EcoRIacbL fragment in pIJ4123 (NdeI/EcoRI)This work pCW4123M1.08-kb NdeI/EcoRIacbM fragment in pIJ4123 (NdeI/EcoRI)This work pMJL11.42-kbacbL PCR fragment in pUC18 (SmaI)This work pMJM11.07-kb acbM PCR fragment in pBluescript SK− (EcoRV)This work pMJN10.86-kbacbN PCR fragment in pBluescript SK (EcoRV)This work pMJN20.86-kbNdeI/BamHI acbN fragment in pET11aP (NdeI/HindIII)This work pMJN50.86-kb NdeI/BamHI acbNin pPWW50 (NdeI/BamHI)This work pMJO10.87-kb acbO PCR fragment in pBluescript SK (EcoRV)This work pMJO70.87-kbNdeI/BglII acbO fragment in pIJ4123 (NdeI/BamHI)This work Open table in a new tab The techniques for all manipulations and the transformation of recombinant DNA molecules and their analysis by restriction and sequencing were performed according to standard protocols or as described earlier (5Stratmann A. Mahmud T. Lee S. Distler J. Floss H.G. Piepersberg W. J. Biol. Chem. 1999; 274: 10889-10896Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 11Hopwood D.A. Bibb M.J. Chater K.F. Kieser T. Bruton C.J. Kieser H.M. Lydiate D.J. Smith C.P. Ward J.M. Schrempf H. Genetic Manipulation of Streptomyces: A Laboratory Manual. John Innes Institute, Norwich, UK1985Google Scholar, 15Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The general strategy for cloning theacbKLMNO genes into the expression vectors indicated in Table I with an N-terminal His-tagged fusion peptide was as follows. The genes were first amplified by PCR from the genomic DNA ofActinoplanes sp. SE50/110 using the primers listed in TableII. The PCR products were then cut by the restriction enzymes for which recognition sites were designed in the respective primer pairs (see Table II) and subsequently introduced by ligation into cut standard vectors (pUC18 or pBluescript II KS), and the inserts were inserted into pET16bP to create reading frames with N-terminal His-tagged fusions. The resulting plasmids were transformed and propagated in E. coli DH5α. The correctness of the nucleotide sequences of the inserts was controlled by DNA sequencing. The resulting His-tagged fusion cassettes were cut out by the enzyme pairs and further ligated to the streptomycete expression vectors given in Table I for later transformation and expression in S. lividans 66 strains. Automated DNA sequencing was carried out on an A.L.F.-Express machine (Amersham Biosciences, Freiburg, Germany) using the Thermosequenase DNA sequencing kit (Amersham Biosciences) and standard primers.Table IIPrimers for amplification of the acb genes investigatedPrimerNucleotide sequence2-aIntroduced recognition sites for restriction endonucleases are underlined.TargetAcbK15′-CAAGGAGACATATGTCGGAGCAC-3′ acbKstartAcbK25′-GTGGTGAGATCTTCGCCCAGT-3′ acbKstop2775M15′-GCCGGCCATATGAAGCGGC-3′ acbMstartpMJM1E5′-CGGTGCCGGTACCACGATCGCGC-3′ acbMstop2775L15′-TTGGTCGGCATATGAGCCGG-3′ acbLstartpMJL.1E5′-GTACGGAATTCGTCCACCGCCAC-3′ acbLstop2775N15′-AGAGGATCACATATGAGCGGGACTC-3′ acbNstartpMJN.1E5′-GAGCTGGATCCCGTC-3′ acbNstop2775O15′-GGTGCGCATATGACCTGCCG-3′ acbOstartpMJO.1E5′-TACCGTCTCGACAGATCTCAGTCAGCTTCCT-3′ acbOstop2-a Introduced recognition sites for restriction endonucleases are underlined. Open table in a new tab Cells were harvested by centrifugation, resuspended in 0.1 volume of disruption buffer (25 mm Tris-HCl, 10 mm MgCl2, 20 mm NH4Cl, and 1 mm β-mercaptoethanol, pH 7.6), and disrupted by sonication (2–3 min at 60 watts). Cell-free extracts were obtained after centrifugation at 13,000 × g for 1 h at 4 °C. The extracts were dialyzed against 5 liters of disruption buffer overnight at 4 °C. The proteins were analyzed by SDS-PAGE as described previously (5Stratmann A. Mahmud T. Lee S. Distler J. Floss H.G. Piepersberg W. J. Biol. Chem. 1999; 274: 10889-10896Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 16Schägger H. van Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10481) Google Scholar). Protein concentrations were determined according to the method of Bradford (17Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar). Generally, the crude extracts as prepared above were tested in assays of 20-μl final volume in a standard buffer system containing 25 mm Tris-HCl, 10 mm MgCl2, 20 mm NH4Cl, and 10 mm2-epi-5-epi-valiolone (or other substrates that were tested) adjusted to pH 7.6. The coenzymes and/or cosubstrates (ATP, NAD, NADH, NADP, NADPH, FAD, or FADH2) were used in final concentrations of 10 mm in the test volumes. The acarbose 7-kinase AcbK was tested as described by Drepper and Pape (18Drepper A. Pape H. J. Antibiot. (Tokyo). 1996; 49: 664-668Crossref PubMed Scopus (13) Google Scholar), but without NH4Cl in the buffer. AcbM tests were routinely performed in a volume of 15 μl. Each assay contained 25 mm Tris-HCl, 10 mm MgCl2, 20 mm NH4Cl, 10 mm ATP, 10 mm2-epi-5-epi-valiolone (or other substrates that were tested) adjusted to pH 7.6, and 12 μl of cell-free extracts. The assays were incubated at 30 °C for 2–12 h. The reaction was monitored by TLC. For radioactive assays, 1 μl of [γ-32P]ATP (2.0 μCi; Amersham Biosciences) was added, and only 11 μl of cell-free extracts were used. Radioactively labeled spots were visualized after TLC by autoradiography with x-ray films (Hyperfilm, Amersham Biosciences). For assaying the activity of AcbO, a mixture of cells from S. lividans/pCW4123M and S. lividans/pMJO7 (1:1) was used. Cell extracts were prepared as described for the AcbM tests. The test conditions and the detection of the reaction product were also identical as for the analysis of the AcbM-catalyzed reaction. Samples of the enzyme reactions were chromatographed on silica thin-layer sheets (Merck, Darmstadt, Germany) using solvent I (isobutyric acid and 1 n NH3 in water, 5:3) or solvent II (butanol/ethanol/water, 9:7:4). The substrates were detected as brown spots after heating or as blue spots after development using a cerium- and molybdate-containing reagent (19Drepper A. Peitzmann R. Pape H. FEBS Lett. 1996; 388: 177-179Crossref PubMed Scopus (17) Google Scholar). 10-ml cell-free extracts from S. lividansTK64/pCW4123M were applied to an Ni2+-HiTrap chelating column (Amersham Biosciences). The column was first washed with 10–20 ml of starting buffer (20 mmNa3PO4, 500 mm NaCl, 20 mm NH4Cl, 10 mm imidazole, and 1 mm β-mercaptoethanol adjusted to pH 7.5) and then washed with a linear gradient of 10–500 mm imidazole in 10 ml of starting buffer and 10 ml of elution buffer (20 mmNa3PO4, 500 mm NaCl, 20 mm NH4Cl, 500 mm imidazole, and 1 mm β-mercaptoethanol adjusted to pH 7.5). The fractions were analyzed by SDS-PAGE. The His-tagged AcbM protein was eluted at ∼200–300 mm imidazole from the column. The partially purified protein was dialyzed for 24 h against 5 liters of dialysis buffer (25 mm Tris-HCl, 10 mmMgCl2, 20 mm NH4Cl, and 1 mm β-mercaptoethanol adjusted to pH 7.6). The enzyme-catalyzed synthesis of 2-epi-5-epi-valiolone was performed in a coupled assay using transketolase (EC 2.2.1.1), ribose 5-phosphate, and hydroxypyruvate to synthesize the substrate sedo-heptulose 7-phosphate in situ according to the protocol described previously (20Sprenger G.A. Schörken U. Sprenger G. Sahm H. Eur. J. Biochem. 1995; 230: 525-532Crossref PubMed Scopus (108) Google Scholar). The cyclization of sedo-heptulose 7-phosphate was performed with cell-free extracts from S. lividans containing the AcbC protein according to the protocol of Stratmann et al. (5Stratmann A. Mahmud T. Lee S. Distler J. Floss H.G. Piepersberg W. J. Biol. Chem. 1999; 274: 10889-10896Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) under the following specific conditions, The preparative enzyme reaction was performed overnight at 30 °C in a total volume of 30 ml. The assay contained 2 units of transketolase (Sigma, Munich, Germany), 10 mmhydroxypyruvate (Sigma), 10 mm ribose 5-phosphate (Sigma), 0.5 mm thiamin pyrophosphate (Sigma), 1 mmMgCl2, 0.025 mm CoCl2, 2 mm NaF, pH 7.6, and variable amounts of cell-free extracts from strain S. lividans 1326/pAS8/7 containing overproduced AcbC protein (1Truscheit E. Frommer W. Junge B. Müller L. Schmidt D.D. Wingeder W. Angew. Chem. Int. Ed. Engl. 1981; 20: 744-761Crossref Scopus (567) Google Scholar). The reaction was monitored by TLC. The product of the AcbC reaction had the same R F value (R F = 0.53, solvent II) as the chemically synthesized 2-epi-5-epi-valiolone ((5R,2S,3S,4S)-5-(hydroxymethyl)cyclohexanone-2,3,4,5-tetrol). The chemical synthesis of racemic 2-epi-5-epi-valiolone was performed according to a new protocol, the details of which will be published elsewhere. 1O. Block and H.-J. Altenbach, unpublished data. 30 ml of the AcbC reaction solution were heated at 90 °C for 5 min, centrifuged (5000 rpm, 20 min), and then applied to an ultrafiltration cell with a YM-10 ultrafiltration membrane (10,000-Da cutoff; Amicon, Witten, Germany). The flow-through was collected. After freeze-drying, ∼250 mg of yellow powder were acquired. The product was dissolved in 3 ml of Milli-Q water and then subjected to anion-exchange chromatography with Dowex 1-X8 (Cl− form, mesh 100–200; Serva, Heidelberg, Germany) on an SR25/50 column (Amersham Biosciences). The column was washed with water, and the fractions containing 2-epi-5-epi-valiolone were pooled. After lyophilization, 20 mg of 2-epi-5-epi-valiolone were obtained as a light-yellow powder. The partially purified AcbM protein was used in phosphorylation assays with 20 mg of purified 2-epi-5-epi-valiolone. The reaction mixture was applied to an Amicon ultrafiltration cell with a YM-10 ultrafiltration membrane (10,000-Da cutoff). The 10-ml flow-through was collected, concentrated to 3 ml by freeze-drying, and then subjected to anion-exchange chromatography with Dowex 1-X8 (Cl− form, mesh 100–200) on a SR25/50 column. The column was washed with plenty of water, and the 2-epi-5-epi-valiolone phosphate was eluted with a linear gradient of 0–600 mm NaCl at a flow rate of 2 ml/min. The elutions were collected as 2-ml fractions and analyzed by TLC. Fractions containing the desired product (total volume of 48 ml) were pooled and concentrated 10-fold by freeze-drying. Desalting the product was carried out at 4 °C on Sephadex G-10 (5.0 × 81-cm SR25/100 column, Amersham Biosciences). The product was eluted with water at a flow rate of 1.5 ml/min. The fractions containing 2-epi-5-epi-valiolone phosphate were pooled. After lyophilization, 12 mg of 2-epi-5-epi-valiolone phosphate were obtained as a white powder. All NMR spectra were recorded on a Bruker ARX 400 spectrometer (400 MHz). In addition to 1H, 13C, and 31P experiments, also COSY (1H-1H, 1H-13C, and 1H-31P) and distortionless enhancement of polarization transfer (DEPT) spectra for the unequivocal correlation of the hydrogen, carbon, and phosphor atoms were recorded. The chemical shifts are given in ppm, related to the solvents as internal standard. The multiplicity is given by the following symbols: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), Ψt (pseudotriplet for unresolved dd), and br (broad). The coupling constant J is given in Hz. The NMR spectra for 2-epi-5-epi-valiolone are as follows:1H NMR (d 4-MeOH, 400 MHz): δ = 2.33 (dd, 1H, J = 13.7, 1.7 Hz, H-6ax), 2.84 (d, 1H, J = 13.7 Hz, H-6eq), 3.43 (d, 1H, J = 11.3 Hz, H-7a), 3.64 (d, 1H, J= 11.3 Hz, H-7b), 4.03 (m, 1H, H-4), 4.27 (Ψt, 1H, J= 4 Hz, H-3), and 4.59 (d, J = 4.0 Hz, H-2);13C NMR (d 4-MeOH, 101 MHz): δ = 46.0 (C-6), 67.7 (C-7), 70.9, 76.1, 79.7 (C-2, C-3, C-4), 81.5 (C-5), and 209.8 (C-1). The NMR spectra for 2-epi-5-epi-valiolone 7-phosphate are as follows:1H NMR (D2O, 400 MHz): δ = 2.38 (d, 1H,J = 13.8 Hz, H-6ax), 2.89 (d, 1H,J = 14.2 Hz, H-6eq), 3.58 (dd, 1H,J = 6.6, 11.7 Hz, H-7a), 3.99 (dd, 1H,J = 9.4, 11.5 Hz, H-7b), 4.18 (m, 1H, H-4), 4.38 (Ψt, 1H, J = 3.8 Hz, H-3), and 4.71 (d, under HDO,J = 4.0 Hz, H-2); 13C NMR (D2O, 101 MHz): δ = 46.38 (C-6), 70.17 (d, J = 5.1 Hz, C-7), 71.19 (C-4), 76.74 (C-2), 79.62 (C-3), 82.86 (C-5), and 101.23 (C-1); 31P{1H} NMR (D2O, 162 MHz): δ = 5.55 (PC-7). The optical rotation of 2-epi-5-epi-valiolone 7-phosphate was [α]D20 + 4.9° (C, 0.35, in H2O). The chromatographic part consisted of a Dionex DX-500 ion chromatography system equipped with a gradient pump (GP40), an eluent generator (EG40) with an EGC-KOH cartridge, a 25-μl injection loop, and an electrochemical conductivity" @default.
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• W2024459080 title "Biosynthesis of the C7-cyclitol Moiety of Acarbose inActinoplanes Species SE50/110" @default.
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