An extracellular enzyme activity in the culture supernatant of the acarbose producer Actinoplanes sp. strain SE50 catalyzes the transfer of the acarviosyl moiety of acarbose to malto-oligosaccharides. This acarviosyl transferase (ATase) is encoded by a gene, acbD, in the putative biosynthetic gene cluster for the alpha-glucosidase inhibitor acarbose. The acbD gene was cloned and heterologously produced in Streptomyces lividans TK23. The recombinant protein was analyzed by enzyme assays. The AcbD protein (724 amino acids) displays all of the features of extracellular alpha-glucosidases and/or transglycosylases of the alpha-amylase family and exhibits the highest similarities to several cyclodextrin glucanotransferases (CGTases). However, AcbD had neither alpha-amylase nor CGTase activity. The AcbD protein was purified to homogeneity, and it was identified by partial protein sequencing of tryptic peptides. AcbD had an apparent molecular mass of 76 kDa and an isoelectric point of 5.0 and required Ca(2+) ions for activity. The enzyme displayed maximal activity at 30 degrees C and between pH 6.2 and 6.9. The K(m) values of the ATase for acarbose (donor substrate) and maltose (acceptor substrate) are 0.65 and 0.96 mM, respectively. A wide range of additional donor and acceptor substrates were determined for the enzyme. Acceptors revealed a structural requirement for glucose-analogous structures conserving only the overall stereochemistry, except for the anomeric C atom, and the hydroxyl groups at positions 2, 3, and 4 of D-glucose. We discuss here the function of the enzyme in the extracellular formation of the series of acarbose-homologous compounds produced by Actinoplanes sp. strain SE50.
Viral Nervous Necrosis (VNN) causes high mortality and reduced growth in farmed European sea bass (Dicentrarchus labrax) in the Mediterranean. In the current studies, we tested a novel Pichia-produced virus-like particle (VLP) vaccine against VNN in European sea bass, caused by the betanodavirus “Red-Spotted Grouper Nervous Necrosis Virus” (RGNNV). European sea bass were immunized with a VLP-based vaccine formulated with different concentrations of antigen and with or without adjuvant. Antibody response was evaluated by ELISA and serum neutralization. The efficacy of these VLP-vaccine formulations was evaluated by an intramuscular challenge with RGNNV at different time points (1, 2 and 10 months post-vaccination) and both dead and surviving fish were sampled to evaluate the level of viable virus in the brain. The VLP-based vaccines induced an effective protective immunity against experimental infection at 2 months post-vaccination, and even to some degree at 10 months post-vaccination. Furthermore, the vaccine formulations triggered a dose-dependent response in neutralizing antibodies. Serologic response and clinical efficacy, measured as relative percent survival (RPS), seem to be correlated with the administered dose, although for the individual fish, a high titer of neutralizing antibodies prior to challenge was not always enough to protect against disease. The efficacy of the VLP vaccine could not be improved by formulation with a water-in-oil (W/O) adjuvant. The developed RGNNV-VLPs show a promising effect as a vaccine candidate, even without adjuvant, to protect sea bass against disease caused by RGNNV. However, detection of virus in vaccinated survivors means that it cannot be ruled out that survivors can transmit the virus.
The putative biosynthetic gene cluster for the α-glucosidase inhibitor acarbose was identified in the producerActinoplanes sp. 50/110 by cloning a DNA segment containing the conserved gene for dTDP-d-glucose 4,6-dehydratase,acbB. The two flanking genes were acbA(dTDP-d-glucose synthase) and acbC, encoding a protein with significant similarity to 3-dehydroquinate synthases (AroB proteins). The acbC gene was overexpressed heterologously in Streptomyces lividans 66, and the product was shown to be a C7-cyclitol synthase using sedo-heptulose 7-phosphate, but not ido-heptulose 7-phosphate, as its substrate. The cyclization product, 2-epi-5-epi-valiolone ((2S,3S,4S,5R)-5-(hydroxymethyl)cyclohexanon-2,3,4,5-tetrol), is a precursor of the valienamine moiety of acarbose. A possible five-step reaction mechanism is proposed for the cyclization reaction catalyzed by AcbC based on the recent analysis of the three-dimensional structure of a eukaryotic 3-dehydroquinate synthase domain (Carpenter, E. P., Hawkins, A. R., Frost, J. W., and Brown, K. A. (1998) Nature 394, 299–302). The putative biosynthetic gene cluster for the α-glucosidase inhibitor acarbose was identified in the producerActinoplanes sp. 50/110 by cloning a DNA segment containing the conserved gene for dTDP-d-glucose 4,6-dehydratase,acbB. The two flanking genes were acbA(dTDP-d-glucose synthase) and acbC, encoding a protein with significant similarity to 3-dehydroquinate synthases (AroB proteins). The acbC gene was overexpressed heterologously in Streptomyces lividans 66, and the product was shown to be a C7-cyclitol synthase using sedo-heptulose 7-phosphate, but not ido-heptulose 7-phosphate, as its substrate. The cyclization product, 2-epi-5-epi-valiolone ((2S,3S,4S,5R)-5-(hydroxymethyl)cyclohexanon-2,3,4,5-tetrol), is a precursor of the valienamine moiety of acarbose. A possible five-step reaction mechanism is proposed for the cyclization reaction catalyzed by AcbC based on the recent analysis of the three-dimensional structure of a eukaryotic 3-dehydroquinate synthase domain (Carpenter, E. P., Hawkins, A. R., Frost, J. W., and Brown, K. A. (1998) Nature 394, 299–302). 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 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 acarbose-like natural products contain, as a unifying structural feature, a pseudodisaccharide based on the C7-cyclitol valienamine bound via an imino bridge to a hexose derivative, which in acarbose is 4-amino-4,6-dideoxyglucose (cf. Fig. 1). Biosynthetically, these compounds resemble aminoglycoside antibiotics (3Piepersberg W. Distler J. Rehm H.-J. Reed G. Kleinkauf H. von Döhren H. 2nd Ed. Bio/Technology:Products of Secondary Metabolism. 7. VCH Verlagsgesellschaft mbH, Weinheim, Germany1997: 397-488Google Scholar, 4Piepersberg W. Strohl W.R. Bio/Technology of Antibiotics. 2nd Ed. Marcel Dekker, Inc., New York1997: 81-163Google Scholar). Also, the C7-aminocyclitol units are considered to be similar to other C7N units, a common structural motif more frequently observed in bacterial secondary metabolites (5Floss H.G. Nat. Prod. Rep. 1997; 14: 433-452Crossref PubMed Scopus (91) Google Scholar). From the labeling patterns of variously 13C-labeled d-glucoses, fed to cultures of validamycin-producing Streptomyces sp. or to acarbose-producing Actinoplanes sp., it was suggested that the valienamine moiety is derived from a C7-sugar precursor formed in reactions of the pentose phosphate cycle (6Toyokuni T. Jin W.-Z. Rinehart Jr., K.L. J. Am. Chem. Soc. 1987; 109: 3481-3483Crossref Scopus (31) Google Scholar,7Degwert U. van Hülst R. Pape H. Herrold R.E. Beale J.M. Keller P.J. Lee J.P. Floss H.G. J. Antibiot. (Tokyo). 1987; 40: 855-861Crossref PubMed Scopus (63) Google Scholar). The genetics and biochemistry of acarbose biosynthesis have not yet been studied in the producing strains. Only speculations are available on the possible enzymatic mechanism(s) by which the C7-cyclitol unit could be formed. However, the 6-deoxyhexoses are frequent building units or side chains in many actinomycete secondary metabolites and are mostly synthesized via a dTDP-hexose pathway (3Piepersberg W. Distler J. Rehm H.-J. Reed G. Kleinkauf H. von Döhren H. 2nd Ed. Bio/Technology:Products of Secondary Metabolism. 7. VCH Verlagsgesellschaft mbH, Weinheim, Germany1997: 397-488Google Scholar, 8Stockmann M. Piepersberg W. FEMS Microbiol. Lett. 1992; 90: 185-190Crossref Google Scholar). Therefore, we used the highly conserved gene sequences of the dTDP-d-glucose 4,6-dehydratase to probe for related genes in the acarbose producerActinoplanes sp. 50/110. In this way, a gene cluster was isolated that contains several genes putatively involved in the biosynthesis of this natural product. Besides genes for dTDP-6-deoxyhexose formation, such as acbA(dTDP-d-glucose synthase) and acbB (encoding dTDP-d-glucose 4,6-dehydratase), a third gene,acbC, was found that encodes an AroB-like protein (dehydroquinate synthase (DHQS) 1The abbreviations used are: DHQS, dehydroquinate synthase; ESI-MS, electrospray ionization mass spectrometry; kb, kilobase pair(s); PCR, polymerase chain reaction; DAHP, 3-deoxy-d-arabino-heptulosonate 7-phosphate.). The acbCgene was expressed heterologously in Streptomyces lividans, and employing the same reaction conditions as used in in vitro studies on DHQS proteins, its product was shown to be a C7-cyclitol synthase using sedo-heptulose 7-phosphate, but not ido-heptulose 7-phosphate, as a substrate. The bacterial strains and plasmids used in this study are listed in TableI. S. lividans 1326 was used as the host strain for the protein expression experiments. The strain was routinely cultured at 28 °C on SMA agar plates (13Distler J. Klier K. Piendl W. Werbitzki O. Böck A. Kresze G. Piepersberg W. FEMS Microbiol. Lett. 1985; 30: 145-150Crossref Scopus (26) Google Scholar) or in tryptic soy broth liquid medium (9Hopwood 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, Norfold, United Kingdom1985Google Scholar). To maintain the plasmid pIJ6021, these media were supplemented with kanamycin (50 μg/ml). The thiostrepton-inducible expression of the cloned acbC gene in S. lividans was carried out according to Takano et al. (11Takano E. White J. Thompson C.J. Bibb M.J. Gene (Amst.). 1995; 166: 133-137Crossref PubMed Scopus (128) Google Scholar) with the exception that 7.5 μg of thiostrepton/ml of YEME liquid medium (9Hopwood 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, Norfold, United Kingdom1985Google Scholar) was used, and the incubation time after induction was prolonged to 20 h.Actinoplanes sp. chromosomal DNA was prepared by standard procedures (9Hopwood 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, Norfold, United Kingdom1985Google Scholar). Subcloning experiments with Escherichia coliwere performed with the vector pUC18 and the host strain DH5α, which was grown at 37 °C in LB broth or on LB agar plates.Table IBacterial strains and plasmids used in this studyStrain or plasmidProperties or productSource or Ref.Bacterial strainsActinoplanes sp. 50/110AcarboseATCC 31044S. lividans 1326Actinorhodin, prodigiosin9Hopwood 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, Norfold, United Kingdom1985Google ScholarE. coli DH5αF−, j80d,lacZ ΔM15, endA1, recA1,hsdR17, (rk− mk+),supE44, thi-1, λ, gyrA96,relA1, Δ(lacZYA-argF) U16910Hanahan D. J. Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8190) Google ScholarPlasmidspBluescript II KS(−)bla,lacZ-α,StratagenepIJ6021kan,tsr, tipAp,11Takano E. White J. Thompson C.J. Bibb M.J. Gene (Amst.). 1995; 166: 133-137Crossref PubMed Scopus (128) Google ScholarpUC18bla,lacZ-α,12Yanisch-Perron C. Vieira J. Messing J. Gene (Amst.). 1985; 33: 103-119Crossref PubMed Scopus (11465) Google ScholarpAS10.3-kb PCR fragment in pUC18This workpAS22.2-kb BamHI genomic fragment from Actinoplanes sp. in pUC18This workpAS511-kb SstI genomic fragment in pUC18This workpAS613-kb BglII genomic fragment in pBluescript II KS(−)This workpAS8/5.11264-bp1bp, base pair. PCR fragment in pUC18,acbCThis workpAS8/71264-bpNdeI/EcoRI fragment of pAS8/5.1 in pIJ6021,acbCThis worka bp, base pair. Open table in a new tab Restriction enzymes and T4 DNA ligase were purchased from Gibco (Eggenstein, Germany) and used in accordance with the manufacturer's instructions. Agarose gel electrophoresis and DNA manipulations of E. coli were done as described by Sambrook et al. (14Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar); transformations ofE. coli were carried out by the method of Hanahan (10Hanahan D. J. Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8190) Google Scholar). The size fractionation of restriction endonuclease-cleaved chromosomal DNA was done on 12-ml 20% sucrose gradients by centrifugation at 74,200 × g for 15 h at 20 °C. Fractions containing DNA fragments of the expected size were pooled and concentrated by ethanol precipitation. Protoplast preparation and plasmid transformation techniques for S. lividans were performed according to published procedures (9Hopwood 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, Norfold, United Kingdom1985Google Scholar, 15Babcock M.J. Kendrick K.E. J. Bacteriol. 1988; 170: 2802-2808Crossref PubMed Google Scholar). For Southern hybridization, the genomic DNA was immobilized on a Hybond N+ membrane (Amersham Pharmacia Biotech, Braunschweig, Germany). Hybridization was performed at 65–68 °C overnight using nick-translated [32P]dCTP (Amersham Pharmacia Biotech)-labeled DNA fragments as shown in Fig. 2. Stringency washes were done with 2 to 0.1× SSC at 65 °C. Electrospray ionization mass spectrometry (ESI-MS) was carried out on a Perkin-Elmer-Sciex API-3 or a Kratos Profile mass spectrometer, and gas chromatography-mass spectrometry on a Hewlett-Packard 5890 gas chromatograph with a 5971A mass selective detector. Proton NMR spectra were recorded on a Bruker AF 300 NMR spectrometer with a MacNMR 5.5 PCI as the instrument controller and data processor, and 13C NMR spectra on a Bruker AC 400 NMR spectrometer. An ISF-4-V culture shaker (Adolf Kuhner AG, Birsfelden, Switzerland) was used for the fermentation of the acarbose producer. Radioactive samples were counted in Bio-Safe II biodegradable scintillation mixture (Research Products International Corp.) in a Beckman LS 1801 scintillation counter. Two different strategies were used to identify the acarbose biosynthesis gene cluster in the genome ofActinoplanes sp. 1) The strD and strEgenes from Streptomyces griseus (8Stockmann M. Piepersberg W. FEMS Microbiol. Lett. 1992; 90: 185-190Crossref Google Scholar), encoding dTDP-d-glucose synthase and dTDP-d-glucose 4,6-dehydratase, respectively, were used as heterologous probes to identify the equivalent gene(s) in the genomic DNA ofActinoplanes sp. by means of DNA-DNA hybridization experiments. For this purpose, a 0.70-kbEcoRI/BglII fragment, containing most of thestrD gene (16Distler J. Ebert A. Mansouri K. Pissowotzki K. Stockmann M. Piepersberg W. Nucleic Acids Res. 1987; 15: 8041-8056Crossref PubMed Scopus (124) Google Scholar), and a 0.76-kb KpnI fragment, containing most of the strE gene (17Pissowotzki K. Mansouri K. Piepersberg W. Mol. Gen. Genet. 1991; 231: 113-123Crossref PubMed Scopus (100) Google Scholar), radioactively labeled as above, were used. 2) Part of the putative dTDP-d-glucose 4,6-dehydratase, belonging to the acarbose biosynthesis gene cluster in the genome of Actinoplanes sp., was amplified by PCR using genomic DNA as template and primers AS2 (5′-GCCGCCGA(A/G)TCCCATGT(G/C)GAC-3′) and AS5 (5′-CCCGTAGTTGTTGGAGCAGCGGGT-3′). Amplification was performed in a Biometra Personal Cycler using 2.5 units of Taq DNA polymerase (Gibco). The reaction mixtures (100-μl volume) contained 200 ng of chromosomal DNA, 50 pmol of each primer, 0.2 mmdNTPs (Boehringer, Mannheim, Germany), incubation buffer, and 5% dimethylformamide. The following conditions were used for the reaction. The enzyme was added after an initial denaturation for 5 min at 95 °C, followed by 25 cycles (95 °C for 1 min, 54 °C for 30 s, and 72 °C for 30 s) and 72 °C for 5 min (ramping rate of 1 °C/s). The PCR product was cloned into pUC18HincII, resulting in pAS1. The amplified 300-base pair DNA fragment was used as a homologous radioactive probe to identify the corresponding gene in the genome of Actinoplanes sp. by means of DNA-DNA hybridization experiments. Various overlapping restriction fragments from the 10.7-kb SstI and 12.4-kb BglII DNA fragment inserts in pAS5 and pAS6, respectively, were subcloned into pUC18 and sequenced by the dideoxynucleotide chain termination method (18Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52678) Google Scholar) using an AutoRead sequencing kit and an A.L.F DNA sequencer (Amersham Pharmacia Biotech, Freiburg, Germany). The entire sequences of both strands were determined from double-stranded plasmid DNAs prepared by the alkaline lysis method (14Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). A double-stranded nested deletion kit (Amersham Pharmacia Biotech) was used to construct unidirectional deletions in DNA fragments in accordance with the manufacturer's instructions. The DNA sequences were analyzed using DNA-Strider 1.2 (19Marck C. Nucleic Acids Res. 1988; 16: 1829-1836Crossref PubMed Scopus (817) Google Scholar) and BrujeneII sequence analysis software. Homology searches were performed against the EBI, GenBankTM, and SWISSPROT data libraries using BLAST (20Altschul S.F. Madden T.L. Schäffer A.A. Zheng Zhang J.Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59933) Google Scholar) and FASTA 1.4x2 (21Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Crossref PubMed Scopus (9381) Google Scholar) software. The putative C7-cyclitol synthase gene (acbC) was amplified by PCR using genomic DNA as template and primers AS-C1 (5′-AGGGAAGCTCATATGAGTGGTGTCGAG-3′) and AS-C2 (5′-GGTATCGCGCCAAGAATTCCTGGTGGACTG-3′). Primer AS-C1 was designed for the introduction of an NdeI site in place of the natural start codon and for the ability to create a start codon fusion ofacbC into the promoter/ribosome-binding site cassette of expression vector pIJ6021. Primer AS-C2 was designed for the introduction of an EcoRI site 117 base pairs downstream of the acbC stop codon for the ligation of the acbCDNA fragment into pIJ6021 NdeI/EcoRI. PCR was performed as described above, and the following conditions were used for the reaction: an initial denaturation for 5 min at 95 °C and then 25 cycles (95 °C for 1 min, 50 °C for 20 s, and 72 °C for 40 s) and 72 °C for 5 min (ramping rate of 1 °C/s). The PCR product was cloned into pUC18 HincII, resulting in pAS8/5.1. The insert was reisolated from this plasmid by digestion with NdeI/EcoRI and ligated into pIJ6021 NdeI/EcoRI. The resulting derivative (pAS8/7) was transformed into S. lividans 1326. Cells were harvested by centrifugation, washed twice in ice-cold buffer A (20 mmK2HPO4/KH2PO4 (pH 7.5), 0.2 mm NAD+, and 0.5 mmdithiothreitol), and suspended in 2 ml of the same buffer. AcbC production was analyzed by SDS-polyacrylamide gel electrophoresis (22Lugtenberg B. Meijers J. Peters R. van der Hoek P. van Alphen L. FEBS Lett. 1975; 58: 254-258Crossref PubMed Scopus (962) Google Scholar). Protein-containing extracts were lysed by boiling in sample buffer (23Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar) and separated on SDS-polyacrylamide gels containing 11% polyacrylamide. Cells of S. lividans 1326/pAS8/7 were disrupted by sonication in buffer A, and the resulting cell-free extract was clarified by centrifugation (15,000 × g, 30 min, 4 °C). The extract was dialyzed against 2.5 liters of buffer A for 12 h at 4 °C. The protein concentration of the extract was determined using a protein microassay kit (Bio-Rad, Munich, Germany) using bovine serum albumin as the standard protein. The AcbC extract could be stored for 3 months at −20 °C without major loss of activity. Heat inactivation was carried out at 95 °C for 5 min. The enzyme activity was measured by a nonradioactive TLC assay. The enzyme assay (100-μl volume) was performed at 30 °C for 2 h in 20 mm phosphate buffer (pH 7.5) containing 20 μg of protein (AcbC extract), 8 mm sedo-heptulose 7-phosphate orido-heptulose 7-phosphate, 0.04 mmCoCl2, and 2 mm NaF. After incubation, 25-μl samples were chromatographed on silica thin-layer sheets (butanol/ethanol/water, 9:7:4), and the substrate and AcbC reaction product were detected as blue spots with a cer-and molybdate-containing reagent (24Drepper A. Pietzmann R. Pape H. FEBS Lett. 1996; 388: 177-179Crossref PubMed Scopus (17) Google Scholar). The preparative formation of the AcbC-generated product was carried out according to one of two alternative methods. (i) The conversion of 35 mg of sedo-heptulose 7-phosphate (corresponding to 21 mm in the assay) catalyzed by AcbC was carried out in a total volume of 5 ml containing 15 mg of protein (AcbC-containing extract of S. lividans 1326/pAS8/7). The conditions for the assay were as described above. For removal of proteins, the 5-ml extract of the preparative AcbC assay was applied first to Centricon 50 tubes (Amicon, Witten, Germany), followed by a second ultrafiltration through Centricon 10 tubes in accordance with the manufacturer's instructions. An aliquot of the filtrate (3.6 ml) was applied to an anion-exchange column (12.5 × 2.5-cm bed size, Dowex 1-X8, 200–400 mesh, Cl− form; Serva, Heidelberg, Germany). The remaining 1.4 ml of filtrate was stored at −20 °C for further examinations. The column was washed with 75 ml H2O, and the flow-through fraction was collected in 3.5-ml fractions. The AcbC reaction product was detected by the TLC assay described above, and the corresponding fractions were pooled and concentrated by freeze-drying. For the ESI-MS analysis, a sample was further purified by isocratic high pressure liquid chromatography on a Lichrospher RP-select B column with H2O/acetonitrile (98.5:1.5) containing 25 mm ammonium acetate. (ii) Incubation ofsedo-heptulose 7-phosphate with the cell-free extract was conducted with 5 mm substrate, 2 mm NaF, 0.05 mm CoCl2, 1 mm NAD+, and 50 μl of cell-free extract, in a final incubation volume of 100 μl of 25 mm potassium phosphate buffer (pH 7.4). The reaction mixture was incubated at 30 °C for 3 h and then lyophilized to dryness. To the residue was added 300 μl of MeOH, and the mixture was agitated in a Vortex mixer and allowed to stand for 30 min before centrifugation to remove the precipitate. The supernatant was subjected to Sephadex LH-20 column chromatography (50 ml, elution with MeOH) to give the cyclization product (99%). The product of the AcbC reaction had the same R F value (R F = 0.53) in the above TLC system as an authentic sample of 2-epi-5-epi-[6-2H2]valiolone ((2S,3S,4S,5R)-5-(hydroxymethyl)-[6- 2S. Lee, T. Mahmud, I. Tornus, E. Wolf, and H. G. Floss, manuscript in preparation. H2]cyclohexanon-2,3,4,5-tetrol),2different from valiolone ((2R,3S,4S,5S)-5-(hydroxymethyl)cyclohexanon-2,3,4,5-tetrol 3Lee, S., Tornus, I., Dong H., and Gröger, S. (1999) J. Labelled Comp. Radiopharm., in press. (R F = 0.48), 2-epi-[6-2H2]valiolone ((2S,3S,4S,5S)-5-(hydroxymethyl)-[6-2H2]cyclohexanon-2,3,4,5-tetrol2(R F = 0.50), and valienone3(R F = 0.55). ESI-MS: m/z 215 (M + Na)+; 1H NMR (300 MHz, CD3OD) δ 2.35 (dd, J = 2, 14 Hz, 6-Ha), 2.86 (dd,J = 1.5, 14 Hz, 6-Hb), 3.44 (d, J = 11 Hz, 7-Ha), 3.66 (d, J = 11 Hz, 7-Hb), 4.05 (dd,J = 1.5, 4 Hz, 4-H), 4.29 (t, J = 4 Hz, 3-H), and 4.62 (d, J = 4 Hz, 2-H); 13C NMR (75 MHz, CD3OD) δc 46.0 (t, C-6), 67.6 (t, C-7), 70.7 (d, C-4), 76.0 (d, C-2), 79.7 (d, C-3), 81.6 (s, C-5), and 209.8 (s, C-1). Trimethylsilyl derivative: gas chromatography-mass spectrometry,T ret = 11.01 min (same as trimethylsilyl-2-epi-5-epi-valiolone; different from trimethylsilyl-2-epi-valiolone (T ret = 11.06 min)); fragment ions:m/z 276 and 480 (M + 3 trimethylsilyl) (2-epi-5-epi-[6-2H2]valiolone,m/z 278 and 482). d-sedo-[1-13C]Heptulose 7-phosphate (82.5% 13C) was prepared froml-[3-13C]serine (containing a trace ofl-[3-14C]serine to guide the isolation of products) and d-ribose 5-phosphate as described (41Lee S.-S. Kirschning A. Müller M. Way C. Floss H.G. J. Mol. Catal. 1999; (in press)Google Scholar). This material (21.25 mg) was incubated with S. lividans1326/pAS8/7 extract in five 1-ml reaction mixtures to give, after Sephadex LH-20 purification, 12 mg of 2-epi-5-epi-[7-13C]valiolone characterized by 1H NMR and showing the expected strongly enhanced 13C NMR signal for C-7. A 10-mg sample of this was fed to two 60-ml resting cell cultures of Actinoplanes sp. strain SN223/29, and acarbose (6 mg) was isolated and purified following previously described procedures (25Lee S. Sauerbrei B. Niggemann J. Egelkrout E. J. Antibiot. (Tokyo). 1997; 50: 954-960Crossref PubMed Scopus (33) Google Scholar). 13C NMR and ESI-MS analysis of the resulting acarbose showed 2% incorporation of the labeled precursor with 13C enrichment specifically at C-7. d-sedo-[7-14C,7-3H]Heptulose 7-phosphate was prepared from d-[6-14C]- andd-[6-3H]glucose as described (41Lee S.-S. Kirschning A. Müller M. Way C. Floss H.G. J. Mol. Catal. 1999; (in press)Google Scholar). A sample of this material (75 nCi, 3H/14C = 5.5) was incubated with S. lividans 1326/pAS8/7 cell-free extract under the assay conditions described above. Purification of the resulting 2-epi-5-epi-valiolone by preparative TLC as described above gave a product of3H/14C = 3.7. To isolate the putative biosynthetic gene cluster for acarbose from the genomic DNA of Actinoplanes sp. 50/110, we chose the widely used strategy to screen the DNA for 6-deoxyhexose-specific genes, which has been described earlier (8Stockmann M. Piepersberg W. FEMS Microbiol. Lett. 1992; 90: 185-190Crossref Google Scholar). For this purpose, restriction digests of the genomic DNA ofActinoplanes sp. were hybridized with DNA probes taken from the strD and strE genes of S. griseus. The strE probe hybridized weakly but specifically with only one band in all cases, e.g. ∼2.2-kb BamHI, 13-kb BglII, and 11-kb SstI fragments (data not shown). The strD probe did not give a signal at all. Therefore, first a PCR approach was used to clone an ∼300-base pair segment of the gene homologous to strE (pAS1; see "Materials and Methods"). This fragment was used to hybridize against genomic DNA of Actinoplanes sp. variously restricted with single endonucleases and combinations thereof. The result was that hybridization was found only in a single genomic region that was identical to that detected with the strE probe (data not shown). The 300-base pair insert of pAS1 was also used as a specific probe to screen size-fractionated genomic DNA libraries of 2–3-kbBamHI, 10–12-kb SstI, and 12–15-kbBglII fragments, cloned in vectors pUC18 or pBluescript II KS(−), for hybridizing plasmids. In each library, hybridizing plasmids were found that contained overlapping genomic DNA segments; they are called pAS2, pAS5, and pAS6 (Fig. 2;cf. Table I). Sequence analysis of the 2.2-kb BamHI DNA fragment inserted into pAS2 revealed the presence of the full-length reading frame of a dTDP-d-glucose 4,6-dehydratase-encoding gene, calledacbB, and two incomplete additional reading frames, each oriented in opposite direction relative to acbB, which were named acbA and acbC (cf. Fig. 2). The sequences of the acbA and acbC genes were completed by subcloning and sequencing overlapping segments from pAS5 and pAS6 and were found to encode a member of the family of dTDP-d-glucose synthases and a protein related to the AroB family of proteins (3-dehydroquinate synthases) of bacteria, respectively. Protein sequence comparisons revealed that the AcbA protein is more related to the RfbA proteins of enterobacteria (57.8% identity in a 218-amino acid overlap to E. coli RfbA) than to the StrD protein of S. griseus (37.0% identity in a 208-amino acid overlap). In contrast, the neighboring acbB gene encodes a protein clearly more related to the streptomycete homolog StrE (57.9% identity in a 318-amino acid overlap) than to the enterobacterial counterpart RfbB (37.0% identity in a 343-amino acid overlap toE. coli RfbB). This explains why the strD gene did not give a hybridization signal. The deduced sequence of the AcbC protein is only distantly similar to the AroB proteins, which among themselves are more strongly conserved (Fig.3). AcbC shows the highest degree of similarity to the AroB protein of Mycobacterium tuberculosis(26.8% identity in a 340-amino acid overlap), which in turn shows significantly higher similarity to the AroB proteins of other bacteria,e.g. E. coli (40.6% identity in a 345-amino acid overlap), Corynebacterium pseudotuberculosis (50.1% identity in a 353-amino acid overlap), and Bacillus subtilis(36.7% identity in a 341-amino acid overlap). However, the eukaryotic DHQS proteins are more distant, e.g. the DHQS domain of the multifunctional AROM protein of Emericella (formerlyAspergillus) nidulans shows only 26.8% identity (in a 340-amino acid overlap) to AroB of M. tuberculosis and a very similar sequence divergence (26.7% identity in a 315-amino acid overlap) to AcbC. However, the DHQS proteins all have strictly conserved amino acid residues in those positions shown to be involved in catalysis and substrate binding, whereas this is the case only for part of those in AcbC (Ref. 26Carpenter E.P. Hawkins A.R. Frost J.W. Brown K.A. Nature. 1998; 394: 299-302Crossref PubMed Scopus (118) Google Scholar; cf. Fig. 3). This suggested that AcbC and AroB do not have identical functions, but that they catalyze similar reactions. The possible involvement of AcbC in the cyclization of the precursor of the C7-cyclitol moiety of acarbose led us to test the hypothesis that this could be formed from a C7-keto sugar phosphate, such as sedo-heptulose 7-phosphate (6Toyokuni T. Jin W.-Z. Rinehart Jr., K.L. J. Am. Chem. Soc. 1987; 109: 3481-3483Crossref Scopus (31) Google Scholar, 7Degwert U. van Hülst R. Pape H. Herrold R.E. Beale J.M. Keller P.J. Lee J.P. Floss H.G. J. Antibiot. (Tokyo). 1987; 40: 855-861Crossref PubMed Scopus (63) Google Scholar). However, when the conversion of sedo-heptulose 7-phosphate was tested in crude extracts of Actinoplanes sp., using reaction conditions suitable for the AroB-catalyzed reaction, no formation of cyclitols could be detected. We then expressed the AcbC protein heterologously in both E. coli and S. lividans 1326. Overexpression in E. coli under control of the T7 promoter was achieved only in the form of insoluble proteins (data not shown). However, induction of expression by thiostrepton inS. lividans 1326/pAS8/7 under control of thetipAp promoter yielded large quantities of soluble protein (Fig. 4). When the crude extracts from induced cells of S. lividans 1326/pAS8/7 were incubated withsedo-heptulose 7-phosphate in the test system developed for the AroB-catalyzed reaction, a rapid conversion of the substrate occurred. sedo-Heptulose 7-phosphate was converted to a substance migrating much faster in the analytical TLC system employed, indicating loss of the phosphate group (data not shown). Co-chromatography with sedo-heptulose, valiolone, and valienone revealed that none of these comigrated with the reaction product. The diastereomeric substrate ido-heptulose 7-phosphate (41Lee S.-S. Kirschning A. Müller M. Way C. Floss H.G. J. Mol. Catal. 1999; (in press)Google Scholar) was not converted under the same conditions. Also, induced extracts from S. lividans 1326/pIJ6021 (control) or heat-inactivated extract (5 min at 95 °C) from S. lividans 1326/pAS8/7 did not cyclize sedo-heptulose 7-phosphate, thereby proving the specificity of the AcbC protein for catalyzing the observed reaction. The initial preparative synthesis and partial purification of the AcbC product yielded a substance, the first NMR and mass spectrometry analyses of which were consistent with its bei
Rifamycin B biosynthesis in Amycolatopsis mediterranei N/813 was inactivated by introducing a small deletion in the rifF gene situated directly downstream of the rifamycin polyketide synthase (PKS) gene cluster. The corresponding mutant strain produced a series of linear intermediates of rifamycin B biosynthesis that are most probably generated by obstruction of the normal release of the end product of the rifamycin PKS. This result provides evidence that the rifF gene product catalyses the release of the completed linear polyketide from module 10 of the PKS and the intramolecular macrocyclic ring closure by formation of an amide bond, as indicated by sequence similarity of this protein to amide synthases. The chemical structures of the new rifamycin polyketide synthase intermediates released from modules 4 to 10 were determined by spectroscopic methods (UV, IR, NMR and MS) and gave insight into the reaction steps of rifamycin ansa chain biosynthesis and the timing of the formation of the naphthoquinone ring. The intermediates released from modules 6 and 8 were isolated as lactones formed by the terminal carboxyl group; proton NMR double resonance and ROESY(rotated frame nuclear Overhauser enhancement spectroscopy) experiments enabled the deduction of the relative configurations in the linear chain which correspond to the known absolute stereochemistry of rifamycin B.
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.
Zielsetzung: Das Verständnis der Signalwege von Lungentumorgeweben ist die Voraussetzung für die Entwicklung neuer und individualisierter Behandlungsstrategien.
