From Chemische Mikrobiologie, FB9-Chemie, Bergische
Universität GH Wuppertal, Gauss-Strasse 20, D-42097 Wuppertal,
Germany and the ¶ Department of Chemistry, University of
Washington, Seattle, Washington 98195-1700
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ABSTRACT |
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The putative biosynthetic gene cluster for the
The -glucosidase inhibitor acarbose was identified in the producer
Actinoplanes 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).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-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
-glucosidases, especially
in the intestine (1, 2). 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
-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 (3, 4). 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 (5). 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 (6, 7).
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Fig. 1.
Chemical structure and biosynthetic building
blocks of acarbose.
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 (3, 8). Therefore, we used the highly conserved gene sequences of the dTDP-D-glucose 4,6-dehydratase to
probe for related genes in the acarbose producer
Actinoplanes 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)1). The acbC
gene 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.
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MATERIALS AND METHODS |
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Bacterial Strains, Plasmids, and Growth Conditions--
The
bacterial strains and plasmids used in this study are listed in Table
I. 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 (13) or in tryptic soy broth liquid medium
(9). 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. (11) with the exception that 7.5 µg of thiostrepton/ml of YEME liquid medium (9) was used, and the
incubation time after induction was prolonged to 20 h. Actinoplanes sp. chromosomal DNA was prepared by standard
procedures (9). Subcloning experiments with Escherichia coli
were performed with the vector pUC18 and the host strain DH5, which
was grown at 37 °C in LB broth or on LB agar plates.
General DNA Manipulation Techniques-- 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. (14); transformations of E. coli were carried out by the method of Hanahan (10). 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 (9, 15). 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.
Instrumentation-- 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.
Strategy for the Identification of the Acarbose Biosynthesis Gene Cluster-- Two different strategies were used to identify the acarbose biosynthesis gene cluster in the genome of Actinoplanes sp. 1) The strD and strE genes from Streptomyces griseus (8), 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 of Actinoplanes sp. by means of DNA-DNA hybridization experiments. For this purpose, a 0.70-kb EcoRI/BglII fragment, containing most of the strD gene (16), and a 0.76-kb KpnI fragment, containing most of the strE gene (17), 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 mM dNTPs (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 pUC18 HincII, 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.
DNA Sequencing and Computer Analysis of Protein and DNA Sequences-- 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 (18) 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 (14). 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 (19) and BrujeneII sequence analysis software. Homology searches were performed against the EBI, GenBankTM, and SWISSPROT data libraries using BLAST (20) and FASTA 1.4x2 (21) software.
Cloning and Expression of the C7-cyclitol Synthase Gene (acbC)-- 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 of acbC 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 acbC DNA 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 mM K2HPO4/KH2PO4 (pH 7.5), 0.2 mM NAD+, and 0.5 mM dithiothreitol), and suspended in 2 ml of the same buffer. AcbC production was analyzed by SDS-polyacrylamide gel electrophoresis (22). Protein-containing extracts were lysed by boiling in sample buffer (23) and separated on SDS-polyacrylamide gels containing 11% polyacrylamide.
Cell Disruption, Enzyme Assay, and Preparative Conversion of
sedo-Heptulose 7-Phosphate Catalyzed by AcbC--
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 or
ido-heptulose 7-phosphate, 0.04 mM
CoCl2, 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 (24). 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 of
sedo-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%).
Characterization of the AcbC Product--
The product of the
AcbC reaction had the same RF value
(RF = 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)-[6-2H2]cyclohexanon-2,3,4,5-tetrol),2
different from valiolone
((2R,3S,4S,5S)-5-(hydroxymethyl)cyclohexanon-2,3,4,5-tetrol3
(RF = 0.48),
2-epi-[6-2H2]valiolone
((2S,3S,4S,5S)-5-(hydroxymethyl)-[6-2H2]cyclohexanon-2,3,4,5-tetrol2
(RF = 0.50), and valienone3
(RF = 0.55). ESI-MS: m/z 215 (M + Na)+; 1H NMR (300 MHz, CD3OD)
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)
c 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, Tret = 11.01 min (same as
trimethylsilyl-2-epi-5-epi-valiolone; different
from trimethylsilyl-2-epi-valiolone
(Tret = 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).
Precursor Role of the AcbC Product and Mechanism of Cyclization-- D-sedo-[1-13C]Heptulose 7-phosphate (82.5% 13C) was prepared from L-[3-13C]serine (containing a trace of L-[3-14C]serine to guide the isolation of products) and D-ribose 5-phosphate as described (41). This material (21.25 mg) was incubated with S. lividans 1326/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 (25). 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]- and
D-[6-3H]glucose as described (41). 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 of
3H/14C = 3.7.
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RESULTS |
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Identification and Partial Cloning of the Acarbose Biosynthesis
Gene Cluster--
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 (8). For
this purpose, restriction digests of the genomic DNA of
Actinoplanes 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-kb
BamHI, 10-12-kb SstI, and 12-15-kb
BglII 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).
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Identification of the Genes acbABC-- 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, called acbB, 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 to E. 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 (formerly
Aspergillus) 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. 26; cf. Fig. 3). This suggested that AcbC
and AroB do not have identical functions, but that they catalyze
similar reactions.
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The C7-cyclitol Synthase AcbC: Overproduction and
Characterization of the Enzyme Reaction--
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 (6, 7).
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 in
S. lividans 1326/pAS8/7 under control of the
tipAp promoter yielded large quantities of soluble protein (Fig. 4). When the crude extracts from
induced cells of S. lividans 1326/pAS8/7 were incubated with
sedo-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 (41) 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.
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Identification of the AcbC Reaction Product--
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 being a valiolone of unknown
stereochemistry. Its RF in the standard TLC system,
between those of synthetic valiolone and valienone, was identical to
that of an authentic sample of synthetic 2-epi-5-epi-[6-2H2]valiolone
((2S,3S,4S,5R)-5-(hydroxymethyl)-[6-2H2]cyclohexanon-2,3,4,5-tetrol),
recently recognized in feeding experiments as the precursor of the
valienamine moiety of acarbose.2 We therefore purified the
AcbC product further and compared it directly with authentic
2-epi-5-epi-[6-2H2]valiolone
by one- and two-dimensional NMR, ESI-MS, and gas chromatography-mass
spectrometry of their trimethylsilyl derivatives. Except for the
predictable spectral differences due to the presence of deuterium in
the standard sample, the two compounds gave identical spectra and
retention times. Incubation of AcbC with a sample of
sedo-[1-13C]heptulose 7-phosphate (41) gave
2-epi-5-epi-[7-13C]valiolone (Fig.
5A), which was fed to a
culture of the acarbose producer, Actinoplanes sp. strain
SN223/29. The resulting sample of acarbose was enriched with
13C at C-7 of the valienamine moiety (Fig. 5C),
confirming the precursor role of the enzymatically generated
2-epi-5-epi-valiolone in acarbose biosynthesis
(Fig. 6). An analogous incubation of
ido-[1-13C]heptulose 7-phosphate with AcbC
again gave no detectable cyclization product.
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Cyclization Mechanism--
Two principal mechanisms are known for
the cyclization of open-chain sugar phosphates to cyclitols. One is
exemplified by the DHQS reaction (27), and the other by the cyclization
of glucose 6-phosphate catalyzed by myo-inositol-1-phosphate
synthase (28). The latter mechanism applied to the synthesis of the
acarbose precursor predicts the loss of both hydrogens from C-7 of the substrate, sedo-heptulose 7-phosphate, whereas in a
DHQS-like cyclization, these two hydrogens would be retained in the
product. We therefore incubated AcbC with a sample of
D-sedo-[7-14C,7-3H]heptulose
7-phosphate and measured the 3H/14C ratio of
substrate and product. The change in this isotope ratio from 5.5 to 3.7 indicates 67% retention of tritium in the cyclization of the substrate
to 2-epi-5-epi-valiolone, a result that rules out
the myo-inositol synthase-like mechanism. The retention of <100% of the tritium is readily explained by nonenzymatic exchange via enolization.
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DISCUSSION |
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The isolation of the putative biosynthetic gene cluster for acarbose from the producer Actinoplanes sp. is hampered by the lack of a genetic system in this organism, reflected by the fact that spore production is very low and a lack of protoplast regeneration and transformation systems. Also, enzymes with direct and specific involvement in the biosynthesis of this type of compounds are unknown so far. Therefore, an approach had to be chosen to isolate genomic DNA of the producing organism directly by means of indicative genetic probes, derived from more widely used genes with likely involvement in this pathway in related organisms. These were found in the genes for the common steps starting the versatile dTDP-hexose pathways used in many secondary metabolic contexts in streptomycetes (3, 8, 29). The two genes acbA, encoding dTDP-D-glucose synthase, and acbB, encoding dTDP-D-glucose 4,6-dehydratase, are members of gene families of widespread use in strain-specific sugar modification pathways and therefore mostly occur in actinomycete secondary metabolic gene clusters or those for the formation of extracellular polysaccharides in Gram-negative bacteria (3, 30, 31). It is interesting to note that both genes and their products are conserved neither in a taxonomic nor in a pairwise coupled fashion, although they are always located in close proximity, often in the same operon, on the DNA (8, 29). No evidence for the existence of more than one copy of a dTDP-D-glucose 4,6-dehydratase gene in the genome of Actinoplanes sp. could be detected, although this is not a rare phenomenon since several bacterial strains, including streptomycetes, have been shown to contain more than one copy of the acbA- and acbB-related gene families, e.g. Streptomyces antibioticus Tü99 (32), E. coli K12 (33), and M. tuberculosis Rv (34) contain two to three copies of both gene families. Nevertheless, the three acb genes exhibit typical actinomycete G + C content (69%) and codon usage (35).
One of the aims of this work, the identification of biosynthetic genes in Actinoplanes sp. for its product acarbose, is accomplished in part since we could demonstrate the enzymatic activity of the AcbC protein to be that of a sedo-heptulose-7-phosphate cyclase producing the acarbose precursor, 2-epi-5-epi-valiolone (see below). The demonstration of this activity in Actinoplanes sp. under acarbose-producing conditions as well as its specific inactivation by insertional mutagenesis in the genome of this organism would be further proofs for the physiological function of acbC, but are not possible at present for the reasons mentioned above. However, the presence of additional genes involved in acarbose metabolism in the neighborhood of the three genes reported here was demonstrated by preliminary DNA sequencing on both sides of the acbABC locus.4 Thus, the genes for the acarbose 7-kinase (acbK) and the acarviosyltransferase (acbD) reported earlier (24, 36) have been detected in close proximity to this subcluster. Therefore, the conclusion is justified that we have identified the gene cluster for acarbose production and metabolism.
The sequence similarity of AcbC to the AroB-related DHQS proteins,
cyclizing 3-deoxy-D-arabino-heptulosonate
7-phosphate (DAHP) to dehydroquinate, suggested that this enzyme
catalyzes C7-cyclitol synthesis using a heptulose
7-phosphate as a substrate in accord with the in vivo
labeling data reported earlier in biogenetic studies on
valienamine-containing metabolites (6, 7). This was clearly proven by
analysis of the enzyme reaction catalyzed by AcbC. Also, the activity
of the AcbC protein is that of a sedo-heptulose-7-phosphate cyclase since it does not cyclize DAHP, nor does, in turn, the E. coli DHQS (AroB), overexpressed in a recombinant strain, cyclize sedo-heptulose
7-phosphate.5 The cyclization
proceeds from sedo-heptulose 7-phosphate, in which C-5, the
carbon undergoing transient oxidation to the ketone, has the same
configuration as in DAHP, opposite to that in the final product,
acarbose, at the same center. It takes place with retention of
configuration at C-5 of the substrate to give a product, 2-epi-5-epi-valiolone, which still must undergo
an epimerization at that carbon (now numbered C-2) during its
conversion into acarbose. The C-5 epimer, ido-heptulose
7-phosphate, which would give the correct acarbose stereochemistry
directly, is not a substrate for AcbC, presumably because its C-5
configuration does not allow oxidation to the ketone. A series of
catalytic events during the cyclization of sedo-heptulose
7-phosphate to 2-epi-5-epi-valiolone is
postulated in Fig. 7 in analogy to the
recent proposal of Carpenter et al. (26). The
stereochemistry of the product at the newly formed stereocenter, the
quaternary carbon C-5, corresponds to that of dehydroquinate, the
cyclization product of DAHP by DHQS. It differs from those of valiolone
and natural valiolamine, which has been isolated from the validamycin
fermentation (37). Another secondary metabolic use of the DHQS
mechanism in actinomycetes seems to occur in the
scyllo-inosose cyclase employed for the first step in the
biosynthesis of 2-deoxystreptamine, a characteristic building block of
a large subclass of the aminoglycosides (3, 38, 39). This enzyme
catalyzes the cyclization of glucose 6-phosphate under equivalent
conditions as the bacterial DHQS enzymes, namely in the presence of
catalytic amounts of NAD+ and cobalt ions and therefore
could represent another member of the same enzyme family. However,
details of the isolation of a gene for a scyllo-inosose
cyclase have not yet been reported, and an internal DNA fragment of
acbC does not hybridize to genomic DNA from actinomycetes
producing streptamine-containing aminoglycosides.4
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Carpenter et al. (26) provided strong evidence that all
catalytic steps occur in the same pocket of the DHQS proteins, based on
their analysis of the three-dimensional structure of the DHQS domain of
the pentafunctional AROM protein of the filamentous fungus
Emericella nidulans. This hypothesis is supported by the fact that the AcbC protein is especially conserved in the putative area
of catalysis and sugar phosphate, cobalt (zinc in the fungal protein)
ion, and NAD+ binding (cf. Fig. 3). Also, 7 of
the 13 amino acid residues of catalytic importance in the DHQS domain
are conserved in AcbC and 4 out of the rest are conservatively
exchanged. Of the catalytically active amino acid residues, just
Lys-152 of DHQS (Glu-162 in AcbC), which is putatively involved in the
phosphate elimination and cyclization steps, and His-275 (Pro-278 in
AcbC), which seems to be engaged in de- and re-protonation of C-5, are
not conserved; however, the function of Lys-152 (DHQS) could also be
complemented by either Arg-161 or Arg-164 in AcbC. Also, the
substrate-binding pocket in the environment of the C-1 and C-2 atoms of
the C7-sugar precursors (formed by, for example, Lys-152,
Lys-250, and Arg-264 in DHQS) seems to deviate widely in AcbC (Ref. 26;
cf. Fig. 3). Therefore, the stereochemical structure of the
product of the AcbC-catalyzed cyclization of sedo-heptulose
7-phosphate (2-epi-5-epi-valiolone) was not a
surprise, although it deviates from the stereochemistry of the cyclitol
moiety at C-2 in the final product, acarbose (cf. Fig. 7).
The AcbC protein clearly belongs to the family of bacterial DHQS (AroB)
proteins, which in contrast to the fungal enzyme, use divalent cobalt
ions as a cofactor instead of zinc ions (40); in the case of the
bacterial DHQS enzymes, zinc ions are even inhibitory. This difference
is quite surprising since the prokaryotic and eukaryotic DHQS proteins
are much more similar to each other than to AcbC, and no suggestive
alterations in the composition of the amino acid residues in the
Co2+ (Zn2+)-binding pocket are evident. The
major differences between AcbC and DHQS proteins are rather to be found
in binding the substrates, which is reflected by the inverted
stereochemistry of the C-4 hydroxyl group and the one carbon extension
at C-2 as well as by the lack of the carbonyl oxygen at C-1 in
sedo-heptulose 7-phosphate relative to DAHP. Therefore, the
cobalt ion and the 3 conserved amino acid residues that are evidently
involved in fixing the C-4 hydroxyl group in the DHQS proteins
(cf. Fig. 3), Asp-146 (Asp-157 in AcbC), Glu-194 (Glu-204),
and Lys-197 (Lys-207), should have slightly different topology in the
substrate-binding pocket of AcbC. Obviously, ido-heptulose
7-phosphate can be discriminated by this protein fold, and
epimerization at C-4 cannot take place. These aspects should be
investigated further, e.g. by amino acid replacement studies
and determination of the crystal structure of the AcbC protein.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y18523.
§ Present address: Pharma, Novartis, CH-4002 Basel, Switzerland.
Present address: Cerep, Inc., Redmond, WA 98052.
** To whom correspondence should be addressed. Tel.: 49-202-439-2521; Fax: 49-202-439-2698; E-mail: piepersb{at}uni-wuppertal.de.
2 S. Lee, T. Mahmud, I. Tornus, E. Wolf, and H. G. Floss, manuscript in preparation.
3 Lee, S., Tornus, I., Dong H., and Gröger, S. (1999) J. Labelled Comp. Radiopharm., in press.
4 A. Stratmann, M. Jarling, P. M. Diaz-Guardamino, H. Thomas, H. Apeler, and W. Piepersberg, unpublished observations.
5 S.-S. Lee and H. G. Floss, unpublished observations.
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ABBREVIATIONS |
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The 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.
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