From the Department of Chemistry, University of Washington, Seattle, Washington 98195-1700
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ABSTRACT |
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The biosynthesis of ansamycin antibiotics, like
rifamycin B, involves formation of 3-amino-5-hydroxybenzoic acid (AHBA)
by a novel variant of the shikimate pathway. AHBA then serves as the
starter unit for the assembly of a polyketide which eventually links
back to the amino group of AHBA to form the macrolactam ring. The
terminal enzyme of AHBA formation, which catalyzes the aromatization of
5-deoxy-5-amino-3-dehydroshikimic acid, has been purified to
homogeneity from Amycolatopsis mediterranei, the encoding gene has been cloned, sequenced, and overexpressed in Escherichia coli. The recombinant enzyme, a (His)6 fusion
protein, as well as the native one, are dimers containing one molecule
of pyridoxal phosphate per subunit. Mechanistic studies showed that the
enzyme-bound pyridoxal phosphate forms a Schiff's base with the amino
group of 5-deoxy-5-amino-3-dehydroshikimic acid and catalyzes both an ,
-dehydration and a stereospecific 1,4-enolization of the
substrate. Inactivation of the gene encoding AHBA synthase in the
A. mediterranei genome results in loss of rifamycin
formation; production of the antibiotic is restored when the mutant is
supplemented with AHBA.
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INTRODUCTION |
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The clinically important ansamycin antibiotic, rifamycin B (Scheme I), contains a biosynthetically unique structural element called a mC7N unit (shown in bold in the rifamycin B structure) (1, 2). This mC7N unit is derived from 3-amino-5-hydroxybenzoic acid (AHBA)1 (3-7), which serves as the starter unit for the assembly of a linear polyketide by addition of acetate and propionate units. The C terminus of the assembled polyketide eventually forms an amide linkage to the amino group of the AHBA moiety to close the macrolactam ring. AHBA, in turn, is generated by a newly discovered biosynthetic reaction sequence, the aminoshikimate pathway, which parallels the first three steps of the shikimate pathway, but is modified by the introduction of nitrogen in the first step (Scheme I) (8, 9) to give 3,4-dideoxy-4-amino-D-arabino-heptulosonic acid 7-phosphate (aminoDAHP) instead of the normal shikimate pathway intermediate, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP). Cyclization and dehydration leads to the 5-amino analog of 3-dehydroshikimic acid, 5-deoxy-5-amino-3-dehydroshikimic acid (aminoDHS), which is then aromatized by the enzyme, AHBA synthase. In this article, we report on the purification and preliminary mechanistic analysis of this enzyme, which has no parallel in the normal shikimate pathway; on the cloning, sequence analysis, and expression of the gene encoding it; and on the effect of deletion of this gene on rifamycin production.
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EXPERIMENTAL PROCEDURES |
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Materials and General Methods--
Fermentation media were
purchased from Difco, and radiochemicals ([-32P]dCTP,
[
-32P]dATP, and [
-35S]dCTP) were from
NEN Life Science Products. AminoDHQ, aminoDHS, and
[7-13C]AHBA (89 atom% 13C) were samples
prepared previously (9). Resins for protein purification and MonoQ and
Phenyl-Superose prepacked FPLC columns were purchased from Pharmacia
Biotech Inc., molecular size standards for SDS-PAGE were from Bio-Rad,
and PVDF membrane from Millipore Corp. All commercially available
enzymes were purchased from Boehringer Mannheim, U. S. Biochemical
Corp., Life Technologies, Inc., or New England Biolabs; the kits for
PCR and sequencing were from U. S. Biochemical Corp.; and
Escherichia coli XL1-Blue and E. coli XL1-Blue
MRF were from Stratagene. BL21(DE3) was obtained from Novagen, and
plasmids were purchased from Novagen (pLysS), Stratagene (pSK
), and
Invitrogen (pRSET). The following buffers were used in the protein
purifications
Purification of AHBA Synthase: Cell Growth-- Amycolatopsis mediterranei S699, a gift from Dr. G. C. Lancini (Lepetit S.A., Geranzano, Italy), was grown as described previously (9). To produce cells for enzyme isolation, 10 ml of a production culture was used to inoculate 500 ml of vegetative medium in a 2-liter flask without a spring coil. After incubation for 54 h at 28 °C and a shaker (New Brunswick Scientific Co., G25) speed of 300 rpm, the mycelia were harvested and used to prepare cell-free extracts.
AHBA Synthase Assay-- AminoDHS solution (500 µl, 0.6 mM) and buffer B were mixed with a portion of the protein solution (50-300 µl) to give a total volume of 1 ml. After 1 h of incubation at 28 °C, the reaction was stopped by the addition of 200 µl of 15% trichloroacetic acid solution. After centrifugation, the production of AHBA was assessed by measuring the increase in A296 relative to a blank. Protein concentration was determined with Bio-Rad protein assay solution, using bovine serum albumin as a standard.
To determine the kinetic parameters of the native or recombinant enzyme, initial velocities were measured by incubating 50 or 100 µl of enzyme solution at 33 °C with 0.1-1.1 mM aminoDHS in buffer C in a total volume of 1 ml and following the absorbance at 310 nm. The reaction was linear for at least 10 min.Ammonium Sulfate Fractionation-- Wet cells of A. mediterranei (25 g) were suspended in 100 ml of buffer B and disrupted by two passages through a French pressure cell. The cellular homogenate was centrifuged at 39,000 × g for 20 min at 4 °C. The supernatant was adjusted to 50% ammonium sulfate saturation by the slow addition, with stirring on ice, of powdered (NH4)2SO4, followed by stirring for an additional 30 min. The precipitate was discarded. The (NH4)2SO4 concentration was then raised to 70%, and the protein pellet was dissolved in a minimal amount of buffer C and dialyzed for 4, 10, and 10 h against three changes of 40 volumes of the same buffer.
Anion Exchange Chromatography-- Preswollen DE 52 resin was equilibrated in buffer C and packed at a bed height of 8 cm (bed volume of 40 ml) following the manufacturer's protocol. The dissolved 50-70% ammonium sulfate precipitate (15-20 ml, 300-350 mg of protein) was applied to the column at a flow rate of 48 ml/h. After the sample had been loaded, the column was washed until the A280 was constant. A linear gradient of 50-350 mM KCl in buffer C (total volume of 240 ml) was applied, and fractions of 4 ml were collected. The fractions containing the AHBA synthase activity (fractions 31-38) were pooled (28-32 ml) and dialyzed overnight against buffer C.
Hydrophobic Interaction Chromatography-- A Phenyl-Sepharose CL-4B suspension in buffer C containing 30% ammonium sulfate was packed in a 2.5 cm × 10-cm column to a bed height of 8 cm (bed volume 40 ml). The sample (28-32 ml, 90-100 mg of protein) from DE 52 chromatography in buffer C 30% saturated with ammonium sulfate was applied to the column at a flow rate of 48 ml/h. The column was then washed with 40 ml of buffer C 30% saturated with ammonium sulfate, followed by elution of AHBA synthase with a linear gradient of 30-0% ammonium sulfate in buffer C (total volume 160 ml). Fractions of 2 ml were collected, and the fractions containing AHBA synthase (fractions 47-55) were pooled (14-16 ml) and dialyzed for 4 and 10 h against two changes of 40 volumes of buffer C.
Gel Filtration-- Sephadex G-200 resin in buffer C containing 150 mM KCl was packed in a column (2.5 cm × 50 cm) at a flow rate of 10 ml/h (bed volume 220 ml). The enzyme solution from the Phenyl-Sepharose column, concentrated to a volume of 2 ml in buffer C containing 150 mM KCl, was applied to the column, which was then eluted with the same buffer at a flow rate of 10 ml/h. Fractions of 3 ml each were collected, and the fractions of high specific activity (fractions 43-48) were combined, concentrated to a volume of 2 ml, and dialyzed for 4 and 10 h against two changes of 40 volumes of buffer C.
Anion Exchange FPLC on Mono Q-- The AHBA synthase solution (8 mg of protein) from the gel filtration was loaded onto a Mono Q column (bed volume 1 ml). The column was washed with buffer C for 5 min at 0.5 ml/min and then eluted with a linear gradient of 50-250 mM KCl in buffer C for 5 min at 1 ml/min. Fractions were collected every 2 min. Step gradients were continued for 10 min with 250 mM KCl and for 15 min with 1 M KCl at a flow rate of 0.5 ml/min. The fractions containing high AHBA synthase activity, eluting just before 250 mM KCl, were pooled.
Hydrophobic Interaction FPLC-- Ammonium sulfate was added to the AHBA synthase pool from the Mono Q FPLC to give 30% saturation. The solution (1.6 mg of protein) was applied to a Phenyl-Superose FPLC column (bed volume of 1 ml) at 0.5 ml/min. The column was flushed with buffer C containing 30% saturated ammonium sulfate for 3 min at 0.5 ml/min, followed by elution with a linear gradient of 30-0% saturated ammonium sulfate in buffer C over 30 min. Fractions were collected every 2 min, and the fractions containing high AHBA synthase activity, eluting at 20-22 min, were pooled. Protein from this step was subjected to SDS-PAGE, and the band corresponding to AHBA synthase was cut out and transferred electrophoretically to Immobilon PVDF transfer membrane (Millipore Corp.). The membrane was used for gas phase microsequencing at the protein analysis facility of the Department of Biochemistry, University of Washington.
DNA Sequencing and Sequence Analysis--
DNA sequencing was
performed using the Sequenase kit (U. S. Biochemical Corp.) and
[-35S]dCTP (NEN Life Science Products) according to
the manufacturer's protocol. Sequencing reactions were analyzed on
polyacrylamide gels (8% (v/v) acrylamide, 5% bisacrylamide, 8 M urea, 45 mM Tris borate, pH 8.0, and 1 mM EDTA). SK, KS, T3, or T7 primers
(Stratagene) for dsDNA sequencing reactions and M13 primer (Stratagene)
for ssDNA sequencing reactions were used. ssDNA was prepared using M13K07 (Promega) following the manufacturer's protocol. DNA and protein sequences were analyzed using the University of Wisconsin Genetics Computer Group (UWGCG) program version 7.3 (10).
Inactivation of AHBA Synthase Gene--
A primary disruption of
the AHBA synthase gene was engineered using a marker-replacement
suicide vector, pSK/AHBA2. In this plasmid, the hygromycin resistance
gene (hyg) of pIJ963 (11), which resides on a 1.75-kb
BglII fragment, had been inserted into the 2.3-kb
XhoI fragment of pSK
/AHBA1 (Fig. 2A) at the
only BglII site, which lies at the N-terminal part of the
AHBA synthase gene, in an orientation such that transcription of the
hyg gene would occur in the same direction as that of the
AHBA synthase gene, leaving 1.2 and 1.1 kb of homologous DNA flanking
this insertion to the left and right, respectively. Through
electroporation (12), approximately 20 transformants of A. mediterranei S699 that were resistant to hygromycin were obtained
per microgram of heat-treated denatured pSK
/AHBA2. Southern
hybridization of the transformants (data not shown) demonstrated that
they arose by the expected single crossover, either upstream, named
HGF001, or downstream, named HGF002, of the AHBA synthase gene, in
approximately equal numbers. HGF001 produced normal rifamycin B yields;
however, all HGF002 recombinants showed delayed production and reduced
yield of rifamycin B (approximately half the yield of wild-type
A. mediterranei S699). One of the HGF001 strains was chosen
for maintenance on synthetic agar medium (13) lacking hygromycin. After
propagation through three subsequent generations, about 0.5-1% of the
colonies showed sporulation 1-2 days earlier and lost the ability to
produce rifamycin B. Southern hybridization with six random choices of these rifamycin B nonproducing colonies (HGF003-1 to -6) (data not
shown) confirmed that pSK
/AHBA2 had integrated into the chromosome in
HGF001, and that all six HGF003 strains had undergone the second crossover event to replace the endogenous AHBA synthase gene with one
that was truncated with the insertion of the hygromycin resistance gene
marker (Fig. 2B).
Expression of the AHBA Synthase Gene--
pSK/AHBA1, which
contains the 2.3-kb XhoI fragment of A. mediterranei DNA carrying the AHBA synthase gene, was digested
with EcoRI. The resulting 1.6-kb EcoRI fragment
was resolved on 0.8% agarose gel and ligated into pRSET digested with
EcoRI using T4 DNA ligase. The ligation products
were transformed into BL21(DE3)/pLysS. Several colonies of E. coli BL21(DE3)/pLysS/pRSET(AHBA) grown on LB agar plates
containing carbenicillin (50 µg/ml) and chloramphenicol (34 µg/ml)
were inoculated into 10 ml of LB medium containing the same
antibiotics. After 5-6 h of growth at 37 °C with shaking (280 rpm),
1 ml of these cultures was used to inoculate 100 ml of LB medium
containing 1 M sorbitol, 2.5 mM betaine, 50 µg/ml carbenicillin, and 34 µg/ml chloramphenicol. These cultures
were incubated at 30 °C until the A600
reached 1.0. Isopropyl-1-thio-
-D-galactopyranoside was
then added to a final concentration of 0.1 mM, and the
expression level during further incubation was checked by SDS-PAGE and
assay of AHBA synthase activity.
Purification of Recombinant AHBA Synthase--
A 500-ml
overnight culture of E. coli BL21(DE3)/pLysS/pRSET(AHBA) in
LB medium containing 1 M sorbitol, 2.5 mM
betaine, carbenicillin, and chloramphenicol
(A600 = 1.7) was transferred to 5 liters of the
same medium in a 10-liter fermentor. Fermentation was carried out at
30 °C and 230 rpm with an aeration rate of 3 liters/min. At an
A600 of 1.2, isopropyl-1-thio--D-galactopyranoside was added to a
final concentration of 0.1 mM. Aeration rate and stirrer speed were increased to 6 liters/min and 280 rpm, respectively. The
cells were harvested after 24 h of growth
(A600 = 1.7) and washed with buffer D.
ESI-MS Analysis of AHBA Synthase-- To a 60-µl sample of purified recombinant AHBA synthase (1 mg of protein/ml, Tris buffer, pH 7.5) were added at room temperature, in 15-min intervals, three portions of a few micrograms each of NaBH4. After 1 h total reaction time, the pH was adjusted to 7.0 by addition of a few microliters of trifluoroacetic acid. The sample as well as a 60-µl sample of unreduced enzyme were desalted by HPLC on a Vydak C-4 column (Rainin) using a linear gradient of water/0.06% trifluoroacetic acid to acetonitrile/0.06% trifluoroacetic acid (1 ml/min over 60 min). The fractions containing the protein (30-35 min) were pooled, lyophilized, and dissolved in 50-60 µl of 60% acetonitrile/40% water/0.06% trifluoroacetic acid. Samples of 20 µl were then introduced into the mass spectrometer at a flow rate of 16 µl/min and data were acquired, averaging over 15-20 scans/sample. Data processing involved the maximum entropy deconvolution (MaxEnt) procedure (14, 15).
Tritiated NaBH4 Reduction of AHBA Synthase-- To 2 mg of purified recombinant AHBA synthase in 0.5 ml of Tris buffer, pH 7.5, was added aminoDHS (1.2 mM) and an excess of tritiated NaBH4 (300 mCi/mmol). After 20 min, the reaction was quenched by addition of 6 N HCl to pH 2. The protein was removed by centrifugation, and the pH was adjusted to 8 with 1 N NaOH. The supernatant, which at this point contained about 23 µCi of tritium, was incubated with alkaline phosphatase and then subjected to preparative layer chromatography (silica gel, n-butanol/water/acetic acid 2:1:1). The majority of the radioactivity coincided with authentic aminoSA (RF = 0.43), the reduction product of the free substrate, but a broad band of radioactivity (~5%) at a higher RF (0.55-0.7) was seen, whereas no radioactivity was evident at the position of authentic 5-deoxy-5-(N-pyridoxylamino)shikimic acid (N-pyridoxyl-aminoSA) (RF = 0.30). The broad high RF band upon rechromatography separated into two bands, one cochromatographing with authentic N-pyridoxyl-AHBA (RF = 0.58) and the other (RF = 0.67) unidentified, but not identical with authentic pyridoxine (RF = 0.44).
A reference sample of N-pyridoxyl-AHBA was synthesized by mixing 7.6 mg of AHBA and 13.3 mg of PLP·H2O, each in 0.5 ml of 0.05 M phosphate buffer, pH 7. After 15 min, when according to TLC Schiff's base formation was complete, excess NaBH4 was added and the mixture kept at room temperature for 1 h with occasional shaking. The product was isolated by ion exchange chromatography (Bio-Rad AG 1X8, formate, successive elution with 3 N HCOOH, 0.1 N HCl, and 1 N HCl) and preparative layer chromatography (silica gel, n-butanol/water/acetic acid 2:1:1, RF = 0.26). The resulting N-phosphopyridoxyl-AHBA was then dephosphorylated by incubation with alkaline phosphatase and the N-pyridoxyl-AHBA purified by preparative layer chromatography in the same system. N-Pyridoxyl-aminoSA was prepared analogously from aminoSA and PLP. ![]() |
RESULTS |
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Enzyme Purification-- AHBA synthase was purified from 54-h-old mycelia of A. mediterranei strain S699 as summarized in Table I. The enzyme activity was assayed by measuring the amount of AHBA formed after 1 h incubation of the protein with 0.3 mM aminoDHS at 28 °C, followed by addition of trichloroacetic acid. In the early stages of purification, AHBA was quantitated by an inverse isotope dilution GC-MS assay (9), in the later stages by measuring the increase in A296, the absorption maximum of AHBA at an acidic pH. The six-step 180-fold purification (Table I) gave AHBA synthase of a specific activity of 72 units/mg protein in 5% overall yield. The protein at this stage was judged by SDS-PAGE to be homogeneous (Fig. 1).
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Properties-- The native molecular mass of AHBA synthase was estimated by gel filtration as 74 kDa and by nondenaturing PAGE as slightly higher than that of bovine serum albumin (66,700 Da). Elution of the enzymatically active band from nondenaturing PAGE and reanalysis by SDS-PAGE gave a molecular mass of 39 kDa, suggesting that the native enzyme is a dimer. Kinetic parameters were determined using partially purified enzyme from the DE 52 column step, assaying activity by following the change in A310 during the initial 10-min linear phase of the reaction. The enzyme is most active at 33 °C and has a pH optimum of 7.5. AHBA synthase retained its activity over a broad range of temperature and pH. Over 84% of the maximum activity of AHBA synthase was maintained over a temperature range from 28 to 50 °C and a pH range from 7.0 to 9.0. The Km value for aminoDHS was determined from Lineweaver-Burk plots as 0.164 mM.
Several compounds were tested as substrates of AHBA synthase, using the protein solution from the Mono Q column step. AHBA synthase could not utilize 5-deoxy-5-amino-3-dehydroquinic acid (aminoDHQ), 5-deoxy-5-aminoshikimic acid (aminoSA), quinic acid, 3-dehydroquinic acid, or 3-dehydroshikimic acid (DHS) as substrate. Furthermore, when the substrate, aminoDHS (0.5 mM), was incubated with AHBA synthase and 3,4-dideoxy-4-amino-D-arabino-heptulosonic acid 7-phosphate (aminoDAHP, 1 mM), aminoSA (1 mM) or 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP, 1 mM), none of the compounds except DAHP affected the enzyme activity, with DAHP showing 40 to 50% activation of the enzyme. It had been shown by others (16) that DAHP synthase from A. mediterranei is inhibited by rifamycin. Activation of AHBA synthase by DAHP provides another piece of evidence that the AHBA biosynthetic pathway is related to the shikimate pathway, and suggests some type of cross-regulation of the two pathways.Partial Amino Acid Sequence Analysis-- To obtain partial amino acid sequence information for the construction of oligodeoxynucleotide primers for the cloning of the AHBA synthase gene, the protein from the Phenyl-Superose FPLC step was further purified by SDS-PAGE. The specific region containing the AHBA synthase band was cut out from the gel after visualization with Coomassie Blue and electrophoretically transferred to PVDF membrane (17, 18). Gas-phase microsequencing of the intact protein revealed a 10-amino acid N-terminal sequence H2N-N-A-R-K-A-P-E-F-P-A (sequence 1). Internal amino acid sequences were determined after in situ CNBr cleavage (19, 20) of the enzyme on the PVDF membrane. The cleavage products were separated by reverse-phase HPLC (21) on a C4 column, and two peptides were chosen for amino acid sequence analysis. They yielded sequences of 26 and 16 amino acids, R-L-N-E-F-S-A-S-V-L-R-A-Q-L-A-R-L-D-E-Q-I-A-V-R-L-E (sequence 2) and G-V-G-P-G-T-E-V-I-V-P-A-F-T-*-I-S (sequence 3).
Cloning of AHBA Synthase Gene-- Based on the above partial amino acid sequences of AHBA synthase, three degenerate oligodeoxynucleotides were designed (Table II), taking into account the preferred codon usage (22) of genes from organisms with GC-rich DNA, like A. mediterranei or Streptomyces (>90% G or C in the third base). These oligonucleotides were used as PCR primers to amplify a 500-bp (combination primers 1 and 2) and a 250-bp (combination primers 1 and 3) region of genomic DNA from A. mediterranei S699. Both PCR products hybridized strongly to the same bands of restriction-digested genomic DNA, establishing them to originate from the same region. Sequencing of the two PCR products revealed that the deduced amino acid sequence of the 257-bp PCR product contained the last two amino acids of the N-terminal peptide sequence and the first six amino acids of the internal peptide sequence 3, neither of which had been used in the construction of the primers. On the other hand, the deduced amino acid sequence of the 505-bp PCR product contained the first 13 amino acids of the internal peptide sequence 2, which had not been encoded by the primer, but did not have the N-terminal amino acid sequence corresponding to oligonucleotide 1 at the 5' end of the PCR product. Instead, the 505-bp PCR product also had the same sequence as the oligonucleotide 2 at the 5' end, suggesting that oligonucleotide 1 might bind nonspecifically within a region where the AHBA synthase gene is located. Furthermore, none of the deduced amino acid sequence corresponding to the peptide 3 was found in the 505-bp PCR product.
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Sequencing and Sequence Analysis of the AHBA Synthase
Gene--
The 2.3-kb XhoI fragment was subcloned into
XhoI-digested pSK. Two white colonies were selected, in
which the 2.3-kb XhoI fragment was present in two different
orientations. A detailed restriction map was obtained (Fig.
2A) using several restriction enzymes, including EcoRI, PstI, SmaI,
SacI, and SalI. XhoI/EcoRI (~800 bp), EcoRI/SacI (~1 kb), and
SmaI/XhoI (~700 bp) digests of the 2.3-kb
fragment were subcloned into pSK
or M13mp18 and sequenced using
pSK
, M13mp18, or sequence-specific primers. The nucleotide sequence
of the AHBA synthase gene was determined in both directions, and the
restriction sites used for subcloning were verified by determination as
part of an overlapping sequence.
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Involvement of AHBA Synthase Gene in Rifamycin
Biosynthesis--
The isolation and characterization of the gene
encoding AHBA synthase reported above sets the stage for the cloning of
the rifamycin biosynthetic gene cluster based on the paradigm (52) that
in Actinomycetes the genes encoding the biosynthesis of a given
antibiotic are clustered in the genome of the producing organism. Thus,
analysis of the DNA surrounding the AHBA synthase gene should reveal
other rifamycin biosynthesis genes, provided that the AHBA synthase
gene is indeed essential for rifamycin formation. To verify this point,
we carried out a gene disruption experiment. An inactivated version of
the AHBA synthase gene was constructed by insertion of a 1.7-kb
BglII hyg-containing DNA fragment from pIJ963
(11) into the unique BglII site in pSK/AHBA1 (Fig.
2A). To reduce the restriction limitation and increase the integration efficiency, this suicide vector was denatured and then
introduced into A. mediterranei S699 by electroporation. Successive selection first for single-crossover mutants
(Hygr) and then for a second crossover gave a
double-crossover mutant (Hygr) (Fig. 2B) in
which the functional AHBA synthase gene had been replaced by the
inactivated version. This mutant was unable to produce rifamycin B, the
normal metabolite of the wild-type, but production was restored to
wild-type levels by supplementation of the culture with AHBA. When the
mutant culture was supplemented with [7-13C]AHBA (89%
13C), the resulting rifamycin B contained at least 81%
13C, confirming the absence of endogenous AHBA synthesis in
the mutant. Thus, the cloned AHBA synthase gene is indeed essential for
rifamycin formation, justifying the expectation that it is part of the
rifamycin biosynthesis gene cluster of A. mediterranei.
Expression of the AHBA Synthase Gene in E. coli--
To establish
whether AHBA synthase is indeed a PLP enzyme and to generate larger
amounts of enzyme for mechanistic studies, the AHBA synthase gene was
overexpressed in E. coli. An EcoRI restriction
digest of pSK/AHBA1 containing the 2.3-kb XhoI restriction fragment produced a 1.6-kb EcoRI fragment in which 23 nucleotides from the start of the coding region of the AHBA synthase
gene had been deleted. The 1.6-kb EcoRI fragment was cloned
into the expression vector pRSET for expression as a (His)6
fusion protein under the control of the T7 promoter. The
cloned vector pRSET(AHBA) (Fig. 2C) was transformed into the
host E. coli BL21(DE3)/pLysS.
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Properties of Recombinant AHBA Synthase-- Although the recombinant enzyme was obtained as a fusion protein and lacked the first six N-terminal amino acids, it showed very similar behavior to the enzyme isolated from A. mediterranei. The native molecular mass of the recombinant AHBA synthase was determined as 80 kDa and that of the denatured protein as 44 kDa by gel-filtration and SDS-PAGE. These values are somewhat low, compared with the molecular mass of 46,101 calculated from the DNA sequence. Nevertheless, the recombinant enzyme, as the native one, is most likely a dimer with subunits of identical molecular mass. The Km value of the recombinant enzyme for aminoDHS was 0.133 mM, compared with 0.164 mM for the native enzyme. The temperature and pH optima, 37 °C and 8.5, respectively, also differed only slightly or predictably from those of the native enzyme, 33 °C and 7.5.
The recombinant enzyme displayed an absorbance maximum at 418 nm, typical of a PLP-lysine Schiff's base (53). Reduction with sodium borohydride abolished the enzymatic activity and led to a change in the absorbance maximum to 330 nm, characteristic of PMP derivatives. PLP forms an adduct with cysteine that absorbs at 330 nm, and this property was used to estimate the amount of PLP bound per mole of AHBA synthase holoenzyme (54). Solutions of AHBA synthase were mixed with 200 mM cysteine in 10 mM HCl, and the A330 was measured. By comparison to standard solutions of PLP, the AHBA synthase was found to contain 0.6 ± 0.05 mol of PLP/mol of enzyme subunit. The protein concentration was measured by both the Bradford method and by UV, using the absorption coefficient calculated (55) from the deduced amino acid sequence of AHBA synthase. The low value compared with an expected one of 1 mol/mol of subunit left open the possibility that only one of the two subunits in the dimeric enzyme carries a PLP. This possibility was ruled out by ESI-MS analysis. ESI-MS was run on the NaBH4-reduced and unreduced, then denatured protein and gave an average molecular mass of 46,075 ± 9 Da for the denatured apoenzyme and 46,297 ± 13 Da for the reduced, denatured holoenzyme. The difference, 222 Da, is close to the molecular mass of the phosphopyridoxyl moiety, 231 Da. Each sample gave only a single protein species, whereas in a spectrum of the mixture the two species were clearly resolved, confirming that the holoenzyme must be a symmetrical dimer in which each subunit carries one molecule of PLP.Mechanistic Role of Pyridoxal Phosphate-- To demonstrate a role of PLP in the catalytic mechanism of AHBA synthase, we examined the effect of an inhibitor. Gabaculine (5-amino-1,3-cyclohexadienelcarboxylic acid), a naturally occurring amino acid isolated from Streptomyces toyocaenis (56), is an irreversible inhibitor of many PLP-requiring aminotransferases (57). Rinehart et al. (58) and Ganem (59) had, in fact, suggested that this compound might be an intermediate on the biosynthetic pathway to mC7N units. In view of the structural similarity of gabaculine and aminoDHS, we checked the effect of gabaculine on AHBA synthase. Incubation of the recombinant enzyme with gabaculine at various concentrations led to irreversible inactivation of the enzyme. The inactivated enzyme did not recover activity upon dialysis against buffer, buffer containing PLP, or buffer containing substrate. The irreversible inhibition of AHBA synthase by gabaculine was time-dependent and showed a biphasic pattern in which the initial, more rapid phase was followed by a second phase of slower decline in activity (data not shown). At an inhibitor concentration of 15 µM, the t1/2 (the preincubation time required for loss of 50% of enzyme activity) was about 20 s at 33 °C. The gabaculine-inactivated enzyme showed a UV absorption maximum at 330 nm. These data clearly implicate the enzyme-bound PLP in the catalytic mechanism of the enzyme (Scheme II).
The involvement of a PLP Schiff's base in the catalytic process was further demonstrated by reduction of the enzyme-substrate complex with tritiated sodium borohydride followed by denaturation of the enzyme, acidic hydrolysis of the products and treatment with alkaline phosphatase. The major tritiated product from the reduction of AHBA synthase preincubated with aminoDHS was identified by comparison with an independently synthesized unlabeled sample as 3-(N-pyridoxylamino)-5-hydroxybenzoic acid (N-pyridoxyl-AHBA) (Scheme III). A second, unidentified product was noted; this was not identical with an authentic reference sample of 5-deoxy-5-(N-pyridoxylamino)shikimic acid. The isolation of tritiated N-pyridoxyl-AHBA provides strong support for the intermediacy of Schiff's bases between the bound PLP cofactor and the nitrogen of the substrate, intermediates, and product in the catalytic mechanism. Furthermore, the fact that reduction of the enzyme-substrate complex produced N-pyridoxyl-AHBA suggests that the intermediate Schiff's base(s) predominating at equilibrium must be relatively product-like, i.e. give reduction products at least capable of aromatizing to the AHBA derivative upon acid hydrolysis.
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Stereospecificity of 6-Deprotonation--
The mechanism of AHBA
formation emerging from the above experiments involves dehydration of
aminoDHS by a PLP-catalyzed ,
-elimination (Scheme
IV). A second necessary step is the loss
of a proton from C-6 of aminoDHS in a 1,4-conjugate enolization. This
deprotonation may be enzyme-catalyzed, and hence stereospecific, or it
may occur nonenzymatically, e.g. by spontaneous
aromatization of a dienone released from the enzyme after the
PLP-catalyzed dehydration of aminoDHS. To probe this issue, we
synthesized (6R)- and
(6S)-[6-2H1]aminoDHS from
(6R)- and
(6S)-[6-2H1]shikimic acid (60) by
the same reaction sequence used to prepare the unlabeled compound (9),
i.e. conversion to aminoSA via the epoxide followed by
enzymatic oxidation at C-3 with E. coli shikimate
dehydrogenase. Incubation of these two substrates with AHBA synthase in
a cell-free extract of A. mediterranei followed by
derivatization and GC-MS analysis of the resulting AHBA (9) showed
>80% enrichment of deuterium from the 6R and <5%
enrichment from the 6S isomer. Hence, the 6-deprotonation of
aminoDHS by AHBA synthase is stereospecific, and thus enzyme-catalyzed,
and involves elimination of the pro-6S hydrogen.
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DISCUSSION |
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Following the establishment of a possible pathway for the formation of AHBA, the precursor of the mC7N starter unit of ansamycin antibiotics, by cell-free experiments (9), we decided to focus on AHBA synthase as the first enzyme of this pathway to investigate in detail. Two considerations prompted this choice. 1) AHBA synthase was the only enzyme that seemed unique to this pathway; unlike the other presumed enzymes it has no equivalent in the normal shikimate pathway. Hence, it was expected that the gene encoding it would represent the most unique probe to isolate ansamycin and mitomycin biosynthetic gene clusters. 2) The chemical aromatization of aminoDHS by acid, base, or buffer treatment proceeds in the same way as that of DHS (61, 62), i.e. it produces exclusively protocatechuic acid (63). Since AHBA synthase generates quantitatively AHBA from the same substrate, the enzyme must completely redirect the aromatization chemistry of aminoDHS; the mechanism by which it achieves this was not obvious.
The studies reported here have revealed this mechanism (Scheme IV) by
showing that the enzyme employs PLP catalysis. The genetic evidence
that the enzyme is a PMP or, more likely, PLP enzyme was corroborated
by analysis of the recombinant protein which showed the presence of a
PLP-Schiff's base. NaBH4 reduction experiments confirmed
the involvement of a Schiff's base between the cofactor and the amino
group of the substrate and ruled out a possible alternative mechanism
proceeding through a Schiff's base between a PMP form of the enzyme
and the C-3 carbonyl group of the substrate. The reaction mechanism of
this enzyme thus can be suggested to resemble those of PLP enzymes
catalyzing ,
-elimination reactions in amino acid metabolism, such
as serine dehydratase or tryptophanase, i.e. the initial
enzyme-substrate Schiff's base first undergoes deprotonation at the
-carbon to give a quinoid intermediate, which then ejects the
electronegative
-substituent, possibly after protonation to
make it a better leaving group (64). The other required part of the
reaction, abstraction of a proton from C-6 in a vinylogous enolization,
is also catalyzed by the enzyme, as evidenced by the fact that this
deprotonation is stereospecific. However, it is not evident from the
data whether this step occurs before or after the 4,5-dehydration of
the substrate.
Interestingly, the enzyme fits the general paradigm of PLP enzymes that, with few exceptions, all the group (proton) transfer reactions take place on only one face of the enzyme-substrate complex (65, 66). The proton at C-5, the OH-group at C-4, and the pro-S proton abstracted from C-6 all are located on the same face of the substrate molecule. Although this may well be fortuitous, it is tempting to suggest that it could reflect the operation of a proton recycling mechanism in which the same enzyme base situated, as in other PLP enzymes, on the Si face of the planar PLP-substrate complex successively mediates the deprotonation at C-5, the protonation of the leaving group 4-OH and the deprotonation at C-6 (65, 66). On the other hand, the enzyme may well only catalyze the deprotonations at C-5 and C-6; if the vinylogous enolization precedes abstraction of the C-5 proton, then the resonance energy gained by aromatization should provide enough driving force for the spontaneous ejection of the 4-OH, i.e. the PLP catalysis may be limited to the generation of the resonance-stabilized carbanion at C-5.
In this context, it is interesting to note that the sequence homology
between AHBA synthase and the PLP enzymes catalyzing ,
-elimination and
-replacement reactions is not particularly strong. Rather, AHBA synthase is most closely related to the deduced amino acid sequences of the products of a family of genes (they have
been called the secondary metabolic
aminotransferase or "SMAT" genes; Ref.
44), many of which are found in antibiotic biosynthesis gene clusters,
which contain a typical PLP or PMP binding motif and seem to be
involved primarily in the formation of antibiotic deoxysugar moieties,
but some possibly also in regulatory functions (Refs. 29 and 42;
cf. Refs. 43-45). As the alignments in Figs. 4 and 5 show,
these genes neatly fall into two subgroups. One carries a modified PLP
binding motif with two adjacent histidines in place of the conserved
active site lysine, and most of them also encode a series of cysteines
which represent an iron-sulfur cluster motif (Fig. 5). This group
mostly seems to be involved in 3-deoxygenation reactions of
deoxysugars; it includes ascC, the gene encoding the well
characterized (46-48) enzyme E1 from Y. pseudotuberculosis,
which catalyzes the 3-deoxygenation reaction in the biosynthesis of
ascarylose. Another member of this subgroup, graG, has
recently been shown by inactivation experiments to be involved in the
biosynthesis of the 2,3,6-trideoxysugar,
L-rhodinose.2 The
second subgroup (Fig. 4) encodes a typical PLP binding site and lacks
the iron-sulfur cluster motif. Its members have been suggested to be
involved in transamination reactions leading to amino deoxysugars and
aminocyclitols. Only recently has transaminase activity of one gene
product from this family been demonstrated. The protein expressed from
stsC from Streptomyces griseus was shown to
catalyze the transamination between glutamine and
scyllo-inosose to give scyllo-inosamine, the
precursor of the aminocyclitol moiety of streptomycin (30). As the
phylogenetic tree in Fig. 7 illustrates, these two subgroups clearly represent two separate branches of this
superfamily of genes which diverged some time ago. AHBA synthase not
only shares the PLP, as opposed to the PMP binding motif with the
proposed transaminases, but is in general much more closely related to
the PLP than to the PMP subgroup, although the dehydration chemistry it
catalyzes formally resembles part of the chemistry of E1 more than the
chemistry of transamination. The membership of an enzyme like AHBA
synthase in this subgroup of PLP genes/enzymes suggests that some other
members of this subgroup may also encode enzymes that catalyze
reactions other than transaminations.
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The cloning of the AHBA synthase gene and the demonstration that this gene is essential for rifamycin biosynthesis provides the means for the isolation of gene clusters encoding the biosynthesis of rifamycin as well as other antibiotics biosynthesized from AHBA. Sequence analysis of cosmid clones of A. mediterranei DNA isolated with the AHBA synthase gene as a probe and extended by chromosome walking has revealed numerous other genes involved in rifamycin biosynthesis. Their sequence and functional analysis will be reported in forthcoming publications.3 The AHBA synthase gene from A. mediterranei has also been used, in a collaboration with the group of E. Leistner, Bonn, to clone homologous genes from Streptomyces collinus Tü 1892 presumably involved in the biosynthesis of ansatrienin (mycotrienin) and naphthomycin,4 and from Actinosynnema pretiosum, presumably involved in ansamitocin biosynthesis.5 Similarly, D. Sherman and co-workers have used the AHBA synthase gene from A. mediterranei to clone the mitomycin biosynthetic gene cluster from S. lavendulae.6 The AHBA synthase gene thus represents another useful addition to the growing inventory of strategic gene probes, which can serve to identify biosynthetic pathways for key natural product classes and to isolate the gene clusters encoding them.
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ACKNOWLEDGEMENTS |
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We are greatly indebted to Kenneth Walsh, Hai Le Trong, and Santosh Kumar of the University of Washington Department of Biochemistry for the amino acid sequence analyses of AHBA synthase, including the preparation and purification of the internal peptide fragments. We are also grateful to Bill Howald of the University of Washington Mass Spectrometry Center for advice and assistance with the ESI-MS analyses of the enzyme, to Richard D. Smith of Battelle Pacific Northwest Laboratories, Richland, WA for attempts to obtain mass spectra of dimeric AHBA synthase, and to Giancarlo Lancini of Lepetit S.A., Geranzano, Italy for providing A. mediterranei strain S699.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Research Grant AI 20264.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) U33061.
To whom correspondence should be addressed: Dept. of Chemistry,
Box 35 17 00, University of Washington, Seattle, WA 98195-1700. Tel.:
206-543-0310; Fax: 206-543-8318; E-mail:
floss{at}chem.washington.edu.
1 The abbreviations used are: AHBA, 3-amino-5-hydroxybenzoic acid; aminoDHS, 5-deoxy-5-amino-3-dehydroshikimic acid; PLP, pyridoxal phosphate; aminoDHQ, 5-deoxy-5-amino-3-dehydroquinic acid; aminoSA, 5-deoxy-5-aminoshikimic acid; aminoDAHP, 3,4-dideoxy-4-amino-D-arabino-heptulosonic acid 7-phosphate; DHS, 3-dehydroshikimic acid; DAHP, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate; ESI-MS, electrospray ionization mass spectrometry; ORF, open reading frame; PMP, pyridoxamine phosphate; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; HPLC, high performance liquid chromatography; bp, base pair(s); kb, kilobase pair(s); GC-MS, gas chromatography-mass spectroscopy.
2 D. Tornus and H. G. Floss, unpublished results.
3 P. R. Aúgúst, L. Tang, Y. J. Yoou, S. Ning, R. Müller, T.-W. Yú, M. Taylor, D. Hoffmann, C.-G. King, X. Zhang, C. R. Hútchinson, and H. G. Floss, Chem. Biol., in press.
4 M. Breuer, D. von Bamberg, E. Leistner, and H. G. Floss, unpublished work.
5 D. Hoffmann, E. Leistner, and H. G. Floss, unpublished work.
6 D. Sherman, personal communication.
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REFERENCES |
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