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J. Biol. Chem., Vol. 277, Issue 25, 22853-22862, June 21, 2002
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,,, and
From the Institutes of Chemical Microbiology and
§ Organic Chemistry, Bergische University, Gauss-Strasse 20, D-42097 Wuppertal, Germany and the ¶ Institute of Biotechnology I,
Research Centre Jülich, Stetternicher Forst,
52425 Jülich, Germany
Received for publication, March 12, 2002, and in revised form, April 5, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We have previously demonstrated that
the biosynthesis of the C7-cyclitol, called valienol
(or valienamine), of the The 
-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 acbC
gene lies in a possible transcription unit, acbKLMNOC, cluster encompassing putative biosynthetic genes for cyclitol conversion. All genes were heterologously expressed in strains of
Streptomyces 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 in
S. 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.
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
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 -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 an
N-glycosidic bond. This acarviosyl moiety is primarily responsible for the inhibitory effect on -glucosidases.
Biosynthetically, these compounds resemble aminoglycoside antibiotics
(3, 4). 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 (4). 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 using
sedo-heptulose 7-phosphate as substrate for the production
of 2-epi-5-epi-valiolone (5). Until now, no other intermediate for the biosynthesis of acarbose has been identified.
View larger version (21K):
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Fig. 1.
Chemical structures of two
-glucosidase inhibitors containing
C7-cyclitols. A, acarbose; B,
validamycin A; C, C7-cyclitols. Valienamine is
not shown as an extra structure because it is represented by the
cyclitol moiety of acarbose including the NH2 group at
C-1.
The trehalase inhibitor validamycin A (cf. Fig. 1B) 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) (6, 7). In contrast, similar feeding experiments revealed 2-epi-5-epi-valiolone to be the only precursor that was incorporated into acarbose (8). 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 in
Actinoplanes 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 (3, 4, 9). 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 the
acbM 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.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Bacterial Strains, Plasmids, and Growth Conditions
The bacterial strains and plasmids used in this study are listed in Table I. The following strains of S. 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 (10), yeast extract-malt extract medium (YEME) medium with 10.3 or 34% sucrose (11), or protoplast regeneration medium (SPMR) plates (12); Actinoplanes sp. SE50/110 was cultured in MD50 medium (2). 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, and acbO genes in S. lividans TK64 was carried out according to Takano et al. (13), 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 (14) supplemented with ampicillin (100 mg/liter).
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Cloning, Manipulation, and Sequencing of DNA
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 (5, 11, 15). The general strategy for cloning the
acbKLMNO 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 of
Actinoplanes sp. SE50/110 using the primers listed in Table II. 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 DH5. 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.
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Preparation of Cell Extracts and Overexpression of Proteins in S. lividans
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 
-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 (5, 16). Protein concentrations were determined
according to the method of Bradford (17).
Enzyme Assays
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 mM 2-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.
AcbK-- The acarbose 7-kinase AcbK was tested as described by Drepper and Pape (18), but without NH4Cl in the buffer.
AcbM--
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 mM
2-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
[
-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).
AcbO-- 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.
Thin-layer Chromatography (TLC)
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 (19).
Purification of the His-tagged AcbM Protein
10-ml cell-free extracts from S. lividans
TK64/pCW4123M were applied to an Ni2+-HiTrap chelating
column (Amersham Biosciences). The column was first washed with 10-20
ml of starting buffer (20 mM
Na3PO4, 500 mM NaCl, 20 mM NH4Cl, 10 mM imidazole, and 1 mM -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 mM
Na3PO4, 500 mM NaCl, 20 mM NH4Cl, 500 mM imidazole, and 1 mM -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 mM
MgCl2, 20 mM NH4Cl, and 1 mM -mercaptoethanol adjusted to pH 7.6).
Synthesis of 2-epi-5-epi-Valiolone
Method a-- 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 (20). 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. (5) 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 mM hydroxypyruvate (Sigma), 10 mM ribose 5-phosphate (Sigma), 0.5 mM thiamin pyrophosphate (Sigma), 1 mM MgCl2, 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 (1). The reaction was monitored by TLC. The product of the AcbC reaction had the same RF value (RF = 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).
Method b-- 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.1
Purification of 2-epi-5-epi-Valiolone
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.
Purification of 2-epi-5-epi-Valiolone 7-Phosphate
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.
NMR
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), 
t
(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 (d4-MeOH, 400 MHz): 
= 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 (t, 1H, J = 4 Hz, H-3), and 4.59 (d, J = 4.0 Hz, H-2);
13C NMR (d4-MeOH, 101 MHz): = 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): = 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 (t,
1H, J = 3.8 Hz, H-3), and 4.71 (d, under HDO,
J = 4.0 Hz, H-2); 13C NMR (D2O,
101 MHz): = 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): = 5.55 (PC-7). The optical rotation of 2-epi-5-epi-valiolone 7-phosphate was
[]
Ion Chromatography
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 detector (ED40). Chromatographic separation was performed on a Dionex Ion Pac AS11 column (250 × 2 mm) provided with a guard column (AG11). An anion trap column (ATC-1) was placed in-line with the GP40 pump and the injection valve to remove anionic contaminants from the eluent. Microfiltered (0.2 µm) water with a specific resistance of at least 18 milliohms was used. The gradient was run as shown in Table II. The Dionex ASRS-II self-regenerating chemical suppressor was housed in an LC30 chromatography oven at 35 °C and operated in the external water mode at 100 mA. The samples were analyzed in triplicate at a flow rate of 0.25 ml/min. The following KOH gradient profile was used: 0-2 min at 100 mM, 2-9 min at 1 mM (point of injection), 9-11 min at 1 mM, 11-15 min increasing from 1 to 19 mM, 15-18 min increasing from 19 to 20 mM, 18-25 min increasing from 20 to 100 mM, and 25-26 min at 100 mM.
Mass Spectrometry
The experiments were performed on a PE-Sciex API 2000 triple-quad instrument (Applied Biosystems, Langen, Germany)
equipped with a Turbolon ion spray and heated nebulizer. To
avoid current leakage, the connection tube between the ion
chromatograph and the mass spectrometer had to be grounded
directly behind the ED40 detector. All measurements were made in
the negative scan mode. Hyphenation was realized with the original flow
rate of 0.25 ml/min at a heater temperature of 380 °C and a
Turbolon ion spray voltage of -4000 V. Optimal gas
(N2) pressures were found as follows: nebulizer gas at
30 p.s.i., heater gas at 90 p.s.i., and curtain gas at
45 p.s.i. Especially for mass-reaction-mass (MRM)
measurements, the collision-added dissociation gas rate was set at
3 p.s.i.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Identification of AcbK as the Acarbose 7-Kinase--
The gene
cluster for the synthesis and metabolism of acarbose and related
components of the amylostatin complex of the producer Actinoplanes sp. SE50/110 has been cloned on a single cosmid
and fully sequenced
recently.2 Its function was
proven by heterologous expression from the cosmid in S. lividans TK23, which formed acarbose-related substances and
characteristic enzyme activities involved in acarbose metabolism such
as the extracellular acarviosyltransferase AcbD (28), and the
cytoplasmic acarbose 7-kinase (18, 19). The gene for the acarbose 7-kinase, acbK, was identified in a putative
operon, acbKLMNOC (GenBankTM/EBI
accession number Y18523) (Fig. 2), by the
identity of the first 20 amino acids of the N-terminal peptide sequence
obtained from the purified enzyme by others (18) to that of the deduced AcbK protein sequence. Also, AcbK exhibited significant similarity to
members of the ribokinase family of phosphotransferases (Fig. 3A). Therefore, we constructed
various plasmids for the expression of AcbK in recombinant derivatives
of both E. coli (BL21(DE3)/pLysS/pCW11K, BL21(DE3)/pLysS/pCW16K, and JM109/pCW2072K) and S. lividans (TK23/pCW201KM6) (cf. Table I). In all
host strains, heterologous (over)expression of soluble AcbK protein,
which was active with or without an N-terminal His tag extension, could
be achieved (Fig. 4A). The
host strains lacked any background activity for phosphorylation of
acarbose. The acarbose 7-kinase AcbK did not phosphorylate
2-epi-5-epi-valiolone or any of the other
possible cyclitol precursors tested in this study in both assay systems
used (Table III; cf. Fig.
1C).
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Identification of AcbM as a 2-epi-5-epi-Valiolone Kinase-- The genes acbLMNO, bracketed by the functionally characterized acbK and acbC genes, were among those putative acarbose biosynthetic genes with some likelihood of being involved in the cyclitol pathway because of their location and the similarities of the encoded AcbL, AcbM, and AcbN proteins to known enzymes (cf. Figs. 2 and 3). The proteins AcbN and AcbL exhibited significant similarities to known oxidoreductases, belonging to two different subfamilies of the dehydrogenases with an N-terminal dinucleotide-binding fold, the zinc-dependent and short-chain alcohol dehydrogenases, respectively (data not shown). AcbM exhibited a distant similarity to GlcK from Bacillus subtilis (GenBankTM/EBI accession number P54495) and other glucokinases, being a member of the hexose 6-kinase family of phosphotransferases (Fig. 3B). No data base entries significantly similar to AcbO were found with all methods of similarity search used (BLAST and fasta 3).
It is known that 2-epi-5-epi-valiolone is the first intermediate in the cyclitol pathway of acarbose, leading to valienol or valienamine (5). It was speculated that the first reaction after the formation of 2-epi-5-epi-valiolone might be catalyzed by either a dehydratase or a dehydrogenase, followed by a step catalyzed by an epimerase (5, 9). To determine whether the next conversion step was catalyzed by AcbL, AcbM, AcbN, or AcbO, the corresponding genes (acbLMNO) were cloned separately in various E. coli expression vectors (pET11a, pET11aP, or pET16bP) (Table I). However, when using these plasmids, none of the proteins could be produced as soluble proteins, although a number of variations in the culture conditions had been tested. Therefore, the genes were cloned in suitable streptomycete expression vectors (pUWL201, pPWW50, and pIJ4123) (cf. Table I) and overexpressed in S. lividans strain TK23, TK64, or 1326. All proteins were well expressed especially when the recombinant pIJ4123 derivatives for the production of N-terminal His-tagged fusion proteins were used (Fig. 4, B-D).
Extracts from S. lividans TK23 with pMJN5 (acbN)
and from S. lividans TK64 harboring pCW4123M
(acbM), pCW4123L (acbL), or pMJO7
(acbO) were prepared and used in a number of various enzyme assays (dehydration, reduction, epimerization, or phosphorylation assays), including chemically synthesized cyclitols (cf.
Fig. 1C) and combinations of the coenzymes ATP,
NAD(P)+, NAD(P)H, FAD+, and FADH2.
In these tests, extracts containing a single overproduced Acb protein
and also combinations of the various extracts were analyzed. The
enzyme tests were monitored by TLC and also photometrically. In none of
the assays was a dehydrogenase-, an epimerase-, or a dehydratase-like
activity detected (data not shown). However, when chemically
synthesized 2-epi-5-epi-valiolone was incubated with cell extracts including AcbM and ATP as a cosubstrate, a new
product was observed upon TLC (Fig. 5).
This product was missing in all assays lacking ATP. Therefore, we
assumed that the new product was
2-epi-5-epi-valiolone phosphate. In subsequent
assays using [-32P]ATP, we determined that the new
product was indeed radioactively labeled. This clearly showed that
2-epi-5-epi-valiolone was phosphorylated. We also
tested the phosphorylation of other cyclitols
(2-epi-5-epi-valiolol, 5-epi-valiolol,
1-epi-2-epi-5-epi-valiolol,
1-epi-5-epi-valiolol, 1-epi-valienol,
and valienol) (Fig. 1C and Table III) in assays with AcbM
extracts. In the presence of [-32P]ATP, only in assays
with 2-epi-5-epi-valiolol and
1-epi-2-epi-5-epi-valiolol were weak
radioactively labeled spots detected on the autoradiogram of the TLC
separation. In nonradioactive assays, these spots were not visible,
indicating very low and less specific phosphorylation of these
compounds (Table III). As the chemically synthesized
2-epi-5-epi-valiolone consisted of a racemic
mixture, only ~50% of the substrate used in the assays was converted
(cf. Fig. 5). To overcome this problem, we synthesized
enantiomerically pure 2-epi-5-epi-valiolone
enzymatically. In a coupled assay (see "Materials and Methods"),
2-epi-5-epi-valiolone was synthesized, and the
purified product was incubated with AcbM-containing extracts. In these
assays, the conversion of 2-epi-5-epi-valiolone to the new phosphorylated spot upon TLC was nearly 100%
(cf. Fig. 5). This result clearly demonstrated that the
enzymatically produced 2-epi-5-epi-valiolone was
the substrate for this phosphorylation step.
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Purification and Structural Characterization of Phosphorylated
2-epi-5-epi-Valiolone--
To prepare
2-epi-5-epi-valiolone phosphate from a purified
system and to identify the position of the phosphate, the substrate 2-epi-5-epi-valiolone was enzymatically
synthesized and purified on a preparative scale, and the His-tagged
AcbM enzyme was purified via its affinity for Ni2+-agarose
(see "Materials and Methods") (Fig.
6). These were used in scaled-up
phosphorylation assays. From these assays, the phosphorylated product
was isolated and purified as described under "Materials and
Methods." The product was first characterized by ion
chromatography-mass spectrometry. The ion chromatography-mass
spectrometry data showed that a substance with a molecular mass of 272 Da was generated. This mass corresponds exactly to the calculated mass
for 2-epi-5-epi-valiolone phosphate. Then
1H, 13C, and 31P NMR data for the
purified substance were determined (see "Materials and Methods")
(Fig. 7). In the 1H NMR
spectrum, the coupling pattern of the former hydroxymethyl group
changed significantly, indicating that it had been modified. A
signal in the 31P NMR at 5.55 ppm is indicative of a
phosphate group, and a 1H-31P COSY experiment
proved that the phosphate group sits at the primary position. The
methylene group occurred in the 1H NMR as an ABX spin
system at 3.58 and 3.99 ppm with coupling constants
3JH-7a,P = 6.6 Hz and
3JH-7b,P = 9.4 Hz,
respectively. Interestingly, while dissolving in
D2O, the AB pattern of the ring methylene group was
replaced by two pseudosinglets at 2.40 and 2.78 ppm within 30 min. This phenomenon can be explained by a fast exchange of the methylene protons
by deuterium from the solvent and has also been observed for the
2-epi-5-epi-valiolone system itself (6). In the
13C spectrum, C-7 appears as a doublet at 70.2 ppm with
coupling constant 2JC-7,P = 5.1 Hz.
Instead of the expected carbonyl group, C-1 shows a resonance for a
quaterneric carbon at 101.23 ppm, indicating a hydratization of the
ketone function. From the described results, the purified product of
the AcbM reaction can be unequivocally identified as
2-epi-5-epi-valiolone 7-phosphate, the second
intermediate in the cyclitol branch of the acarbose pathway.
|
|
2-epi-5-epi-Valiolone 7-Phosphate Is the Substrate of AcbO-- To obtain further insight into the biosynthetic pathway for valienol, cell extracts containing AcbM were combined with extracts containing AcbO, AcbL, or AcbN. In all tests, 2-epi-5-epi-valiolone and ATP were used as substrates without or in combination with various dinucleotides as redox or epimerase coenzymes (NAD, NADH, NADP, and NADPH). Only when extracts with AcbM/ATP in combination with AcbO were used was a new spot observed on the TLC plates (Fig. 5). Without AcbM and in the presence of ATP alone, no conversion of 2-epi-5-epi-valiolone by AcbO was monitored. These data give an important hint for AcbO being the next enzyme in cyclitol conversion. In these assays, no dinucleotide coenzyme was required, although extensively dialyzed cell extracts were used. This finding indicated that the reaction catalyzed by AcbO was coenzyme-independent or that if a cofactor was involved, it must have been tightly bound to the enzyme. Therefore, it was further investigated whether AcbO could be either (i) the epimerase that catalyzes the epimerization at C-2 to give 5-epi-valiolone phosphate or (ii) a dehydratase that catalyzes the formation of 2-epi-valienone 7-phosphate.
Characterization of the AcbO Product as a Probable Epimer of
2-epi-5-epi-Valiolone 7-Phosphate--
To identify the conversion
product from 2-epi-5-epi-valiolone 7-phosphate
that was obtained in the reaction catalyzed by AcbO, the ion
chromatography-mass spectrometry data of the reaction product were
analyzed. In assays containing
2-epi-5-epi-valiolone/ATP and the overproduced
enzyme AcbM alone or AcbM and AcbO together, three new mass peaks of
phosphorylated compounds were detected: m/z 253 (13.27 min), 271 (12.77 min; 2:1 ratio), and 287 (12.97 min). These
peaks were missing in assays without AcbM. The dominant mass peak
(m/z 271) corresponded exactly to the expected
mass for 2-epi-5-epi-valiolone 7-phosphate. As
the same mass peak (m/z 271) was detected in
assays with AcbO, we concluded that AcbO most likely catalyzed the
epimerization at C-2, yielding 5-epi-valiolone 7-phosphate.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Earlier work has demonstrated that 2-epi-5-epi-valiolone is the precursor of the C7-cyclitol unit of the acarviosyl moiety of acarbose (5, 8). The expectation was that a series of enzyme-catalyzed steps involving dehydration, reduction, and epimerization converted this precursor to valienol (or valienamine) before its incorporation into the pseudodisaccharide acarviosine. Although a number of feeding experiments with possible intermediates were described, all other steps and intermediates of this biosynthetic route remained unclear so far (8).
In this study, we have shown that, unexpectedly, the second step after initial formation of the cyclitol precursor by the cyclase AcbC represents a modification by phosphorylation. The reaction involves the conversion of 2-epi-5-epi-valiolone to 2-epi-5-epi-valiolone 7-phosphate, which is catalyzed by a novel kinase, AcbM. The AcbM polypeptide chain shows distant similarity to some members of the hexokinase family of sugar phosphotransferases, which form a cluster of orthologs in complete genome sequences (COG1940; www.ncbi.nlm.nih.gov/COG) (30). This similarity is intriguing because both enzymes D-glucose 6-kinase (GlcK) and 2-epi-5-epi-valiolone 7-kinase (AcbM) phosphorylate the primary hydroxyl group on a cyclic polyol with a six-member ring system. This might indicate that not only valienol, but also its precursor 2-epi-5-epi-valiolone, has a distinct structural resemblance to hexoses. The C7-cyclitol unit of acarbose and its precursors behave functionally as sugar-related metabolites, which are metabolized inside cells mainly as their phosphorylated or nuleotidylated forms. The same position of the cyclitol moiety of free acarbose molecules is also phosphorylated inside cells by the acarbose 7-kinase, encoded by the acbK gene in the same putative transcription unit together with acbM and some other genes (cf. Fig. 2) (Refs. 19 and 29 and this work). During tests for the substrate specificity of AcbK, others had measured a weak activity also with two C7-cyclitols not directly related to valienol metabolism and with a derivative of acarbose lacking the valienol moiety (19). These assays had been carried out with an indirect measurement of ATP consumption, and no direct determination of the phosphorylated products was achieved. However, in our hands, AcbK acted only on the oligomeric end product acarbose and not on any of the monomeric C7-cyclitols that are likely precursors of valienol or on valienol itself. In contrast, AcbM seems to phosphorylate a monomeric cyclitol and to be specific only for the first cyclic intermediate of valienol biosynthesis, 2-epi-5-epi-valiolone, but not for any other monomeric C7-cyclitols. Therefore, we conclude that AcbM is a biosynthetic enzyme, whereas AcbK has another, possibly protective and/or transport-prone function(s) (see below).
Further conversion of the cyclitol precursor 2-epi-5-epi-valiolone 7-phosphate is catalyzed by the isomerase AcbO, which is possibly a 2-epimerase. This conversion step was unexpected because it seemed more likely that either dehydration at positions 5 and 6 or reduction of the keto group at position 1 of the cyclitol came first. The fact that 2-epi-5-epi-valiolone is first phosphorylated and only thereafter further modified by other biosynthetic enzymes such as AcbO is in good accordance with all feeding experiments with whole cells that have been performed so far (8). In these studies, 2-epi-5-epi-valiolone was the only extracellularly provided C7-cyclitol that was incorporated into acarbose. In contrast to these results, in addition to the identical starter metabolite 2-epi-5-epi-valiolone, some additional intermediates (5-epi-valiolone and valienone) were incorporated into the trehalase inhibitor validamycin A, although it shares the same cyclitol moiety, valienol (or valienamine) (cf. Fig. 1) (6). Therefore, the biosynthesis of the two C7-cyclitol units (valienamine and validone) seemed to occur without initial phosphorylation by direct epimerization and dehydration or reduction (7). Also, the incorporation of the nitrogen into validamycin must occur on another route because, in acarbose, it is introduced via formation of a dideoxyaminohexose (see below). So, if phosphorylated cyclitol intermediates are used, e.g. before condensation, they occur in a later phase; and as compared with acarbose synthesis in Actinoplanes sp. SE50/110, a completely different pathway seems to be used in the validamycin producer S. hygroscopicus ssp. limoneus.
The 7-phosphorylation of the C7-cyclitol moiety during the
de novo synthesis of acarbose could be necessary to prevent
an inhibitory effect of the C7-cyclitol- and
acarviosyl-containing intracellular metabolites on cytoplasmic enzymes
sensitive to those inhibitors, such as -glucosidases and
glucomaltases of the producer. For instance, after 7-phosporylation of
acarbose by AcbK, the cytoplasmic and acarbose-sensitive maltase
activity of Actinoplanes sp. SE50/110 is no longer strongly
inhibited by the modified inhibitor (19). Therefore, this
phosphorylation can be regarded as a resistance-like self-protection
mechanism similar to those in other aminoglycoside producers (3, 31). It was shown earlier that the 6-phosphorylations of both streptomycin precursors and streptomycin itself in Streptomyces griseus
strains, which also are catalyzed by a biosynthetic 6-kinase (probably StrN) acting on a monomeric cyclitol intermediate
(N-amidinostreptamine) and a resistance-conferring 6-kinase
(AphD), always keep the bioactive metabolites in an inactivated state
intracellularly (reviewed in Refs. 3 and 32). In addition,
(dihydro)streptomycin 6-phosphate seems to be the export form of this
antibiotic. It becomes dephosphorylated to the bioactive form only by a
specific phosphatase (StrK) outside the cell (33). This coupling of
resistance and active export mechanisms seems to be a widespread
strategy used in producers of self-toxic metabolites. Similar modes are
used also in the producers of macrolides (by glucosylation) (34) and
puromycin and phosphinothricin (by acetylation) (35, 36).
Therefore, we speculate that the 7-phosphorylation of acarbose-related
metabolites (the intracellular end product of the underlying pathway
has not yet been identified) has, in addition, a role in their export to the environment.
Acarbose can be regarded both physiologically and structurally as a member of the aminoglycoside group of bacterial products (3-5). For instance, the three subunits of streptomycin, before condensation, are either phosphorylated (streptidine 6-phosphate) or nucleotidylated (dTDP-L-dihydrostreptose or dTDP-L-rhamnose and nucleoside diphosphate-N-methyl-L-glucosamine). Condensation occurs via typical glycosyltransferase-catalyzed reactions. In the new pathway for acarbose, which we present here, the subunits are phosphorylated and/or activated by nucleotidylation and condensed by glycosyl transfer, too. The postulated cyclitol transferase AcbS (related to bacterial glycosyltransferases; COG0297) would catalyze the last step.
From the data presented herein and from the analysis of the whole
acb cluster, we propose a new pathway for the biosynthesis of the acarviosyl unit of acarbose (Fig.
8). The main characteristics of our
suggestion for this biosynthesis are (i) the phosphorylation of
2-epi-5-epi-valiolone at C-7 prior to its
modification; (ii) the further modification of the cyclitol 7-phosphate
intermediate by 2-epimerization, 5,6-dehydration, and 1-reduction,
resulting in 1-epi-valienol 7-phosphate; (iii) a second
phosphorylation and a subsequent nucleotidylation step at C-1 in
1-epi-valienol 7-phosphate; and (iv) the incorporation of
the amino nitrogen into the sugar moiety and not into the cyclitol
unit. Evidence for the phosphorylation of
2-epi-5-epi-valiolone and its further conversion
products has been presented in this work. In additional experiments, we
have found evidence that 1-epi-valienol is phosphorylated by
a crude extract from Actinoplanes sp. SE50/110 (data not
shown); experiments with 1-epi-valienol 7-phosphate as the
more likely substrate (because extracellularly applied
1-epi-valienol is not incorporated) (8) in similar assays
are in progress. Phosphorylation at C-1 would be a prerequisite for a
subsequent nucleotidylation step. In the acb cluster, we
have identified the gene acbR, which encodes a GlgC
(ADP-glucose synthase; COG0448)-like protein, which could
catalyze the nucleotidylation of the likely precursor
1-epi-valienol 1,7-diphosphate. We also have experimental
evidence that the gene acbV encodes a GabT-like
aminotransferase, which uses dTDP-4-keto-6-deoxyglucose and
L-glutamate as substrates for the synthesis of
dTDP-4-amino-4,6-dideoxy-D-glucose.3
From all these data, it seems most likely that the acarviose is the
condensation product of nucleoside
diphosphate-1-epi-valienol 7-phosphate and
dTDP-4-amino-4,6-dideoxy-D-glucose. The putative glycosyltransferase AcbS is postulated to fulfill this function. It is
still unclear whether acarviose (or activated acarviose or acarviose
7-phosphate) or acarbose is the final product that is synthesized
inside the cell. So far, we do not know if the glucose or maltose
moieties of acarbose are introduced inside the cell or after the export
of the activated acarviose. If acarviosylglucose 7-phosphate is the
exported product, the acarviosyltransferase AcbD described by Hemker
et al. (28) could be a candidate that adds, via
transglycosylation, the maltose or maltotriose residues to the
acarviosyl unit outside the cell to form acarbose and the higher order
oligomeric components found in the fermentation broth of
Actinoplanes sp. SE50/110.
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ACKNOWLEDGEMENTS |
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We are grateful to Bayer AG for supporting this project in many respects, especially the great collaboration with researchers from the Biochemical Development Division, particularly A. Crueger and H. Wehlmann. We thank H. G. Floss and H. Pape for many discussions and our collaborators Y. Rockser and E. Sauerbier for expert technical assistance.
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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.
To whom correspondence should be addressed. Tel.:
49-202-439-2521; Fax: 49-202-439-2698; E-mail:
piepersb@uni-wuppertal.de.
Published, JBC Papers in Press, April 5, 2002, DOI 10.1074/jbc.M202375200
1 O. Block and H.-J. Altenbach, unpublished data.
2 H. Thomas, U. F. Wehmeier, A. Stratmann, M. Jarling, and W. Piepersberg, unpublished data.
3 M. Diaz-Guardamino and W. Piepersberg, unpublished data.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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, no phosphorylation.
