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Antimicrobial Agents and Chemotherapy, May 2000, p. 1214-1222, Vol. 44, No. 5
Pharmazeutische Biologie, Pharmazeutisches
Institut, Eberhard-Karls-Universität Tübingen, 72076 Tübingen, Germany
Received 12 August 1999/Returned for modification 9 December
1999/Accepted 29 January 2000
The novobiocin biosynthetic gene cluster from Streptomyces
spheroides NCIB 11891 was cloned by using homologous
deoxynucleoside diphosphate (dNDP)-glucose 4,6-dehydratase gene
fragments as probes. Double-stranded sequencing of 25.6 kb revealed the
presence of 23 putative open reading frames (ORFs), including the gene
for novobiocin resistance, gyrBr, and at least
11 further ORFs to which a possible role in novobiocin biosynthesis
could be assigned. An insertional inactivation experiment with a
dNDP-glucose 4,6-dehydratase fragment resulted in abolishment of
novobiocin production, since biosynthesis of the deoxysugar moiety of
novobiocin was blocked. Heterologous expression of a key enzyme of
novobiocin biosynthesis, i.e., novobiocic acid synthetase, in
Streptomyces lividans TK24 further confirmed the
involvement of the analyzed genes in the biosynthesis of the antibiotic.
Novobiocin is produced by
Streptomyces spheroides and Streptomyces niveus
and belongs to the aminocoumarin antibiotics. Bacterial DNA gyrase
represents the target of these coumarins (41), and novobiocin inhibits this enzyme by interaction with the N-terminal 24-kDa subdomain of the gyrB subunit (27). In
addition to its antibacterial action, novobiocin shows synergistic
effects with antitumor drugs such as etoposide or teniposide (37,
49).
Little is known about the biosynthesis of novobiocin. Structurally, it
is composed of three moieties, a noviose sugar (ring C), a substituted
coumarin (ring B), and a prenylated 4-hydroxybenzoic acid (ring A), and
these rings are linked by glycosidic and amide bonds (Fig.
1). Radioactive feeding experiments in
the 1960s and 1970s showed that noviose is directly derived from
D-glucose, whereas tyrosine serves as a precursor of ring A
and ring B (3, 6, 31). This was recently confirmed by a
feeding experiment with [1-13C]glucose (33)
which also showed that the dimethylallyl moiety of novobiocin was
formed through the nonmevalonate pathway.
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification of the Novobiocin Biosynthetic Gene
Cluster of Streptomyces spheroides NCIB 11891
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (19K):
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FIG. 1.
Hypothetical biosynthetic pathway of novobiocin. Roman
numerals refer to the putative assignments of genes identified in the
biosynthetic reactions (see Table 1). SAM,
S-adenosylmethionine.
Molecular biological studies have been restricted to the investigation of novobiocin resistance genes (43, 52), especially gyrBr (61, 62), and the production of novobiocin-deficient mutants (19). Discovery of the genetic basis of the biosynthesis of aminocoumarin antibiotics could provide a useful tool for drug development. "Combinatorial biosynthesis," the interchange of genes involved in antibiotic biosynthesis between different microorganisms or the creation of hybrid genes and, consequently, proteins with new enzymatic properties, allows the production of modified or even novel antibiotics (23). In the past, much effort has been undertaken in the manipulation of the biosynthesis of polyketide antibiotics (25, 42, 56), and recently, progress has also been made in the construction of hybrid peptide synthetase genes (55, 59). The discovery of gene clusters for other types of secondary metabolites can offer additional possibilities for combinatorial biosynthesis.
Here we report on the identification of the novobiocin biosynthetic gene cluster from S. spheroides NCIB 11891. The gene cluster was analyzed by molecular cloning, DNA sequencing, an insertional inactivation experiment, and heterologous expression of a key enzyme of novobiocin biosynthesis, i.e., novobiocic acid synthetase.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Bacterial strains, plasmids, and culture conditions.
Plasmids pBluescript SK(
) [pBSK()], pBluescriptC SK()
[pBCSK()], and SuperCosI were purchased from Stratagene
(Heidelberg, Germany), Litmus 28 was purchased from New England Biolabs
(Hertfordshire, United Kingdom), and pGEM-11Zf(+) was purchased from
Promega (Madison, Wis.). pBSKT (36) was a gift from J. A. Salas (Oviedo, Spain). Shuttle vectors pEM4 (48) and
pUWL201 were kindly provided by A. Bechthold and were originally
obtained from J. A. Salas (Oviedo, Spain) and W. Piepersberg
(Wuppertal, Germany), respectively.
DNA isolation, manipulation, and cloning. Standard procedures were performed as described by Sambrook et al. (54) and Hopwood et al. (20). Isolation of DNA fragments from agarose gels was carried out with the Qiagen QIAEX II Gel Extraction Kit (Qiagen, Hilden, Germany). Genomic DNA was isolated from Streptomyces strains by lysozyme treatment and phenol-chloroform extraction as described elsewhere (1).
The S. spheroides NCIB 11891 cosmid library was constructed by partial Sau3A digestion of chromosomal S. spheroides DNA and ligation into the BamHI sites of SuperCosI. Packaging was performed with Gigapack II (Stratagene). Southern blot analysis was performed on Hybond-N nylon membranes (Amersham, Braunschweig, Germany) with digoxigenin-labeled probes by using the DIG high prime DNA labeling and detection starter kit II (Boehringer, Mannheim, Germany).DNA sequencing and computer analysis.
For sequencing,
fragments of approximately 200 to 3,000 bp in length were obtained from
cosmid clones 9-6G and 10-9C and were subcloned into pBSK(),
pBCSK(), or Litmus 28. Sequencing was performed by the
dideoxynucleotide chain termination method with M13 primers or primers
that consisted of fragments from within the sequenced region on a
Molecular Dynamics Vistra 725 DNA sequencer or with a LI-COR automatic
sequencer (MWG-Biotech, Ebersberg, Germany). Both strands of the region
between novA and novW (a total of 25,616 bp) were sequenced.
Construction of novobiocic acid synthetase expression plasmids. To determine the genes involved in the novobiocic acid synthetase reaction, several fragments from cosmid 9-6G were cloned into the Streptomyces expression vectors pEM4 and pUWL201, both of which contain the ermE up promoter for foreign gene expression.
(i) pSL3(+). A 9.7-kb EcoRI fragment containing the open reading frames (ORFs) from novH to novN was cloned into the EcoRI site of pEM4. The correct orientation of the insert in regard to the orientation of promoter was confirmed by restriction analysis.
(ii) pMS65. A 1.95-kb EcoRI-BglII fragment, which was obtained from the 9.7-kb EcoRI fragment mentioned above and which contained the novH gene, was cloned into pUWL201 that had been digested with EcoRI and BamHI to give pSM65.
(iii) pMS71.
The 9.7-kb EcoRI fragment from
cosmid 9-6G was cloned into the EcoRI site of pBSK(),
resulting in plasmid p9-6GE9-R; the SpeI site of pBSK()
was located downstream of novN. After deletion of the
fragment containing the genes from novK to novN
by SnaBI and SpeI digestion of p9-6GE9-R, blunt
ends were generated with T4 DNA polymerase (Amersham, Braunschweig,
Germany), and the construct was religated. The insert of the resulting
plasmid, comprising the genes from novH to novJ,
was digested with EcoRI and XbaI and was cloned
into the same sites of pUWL201 to give pMS71.
(iv) pMS75.
The 9.7-kb EcoRI fragment from cosmid
9-6G was cloned into the EcoRI site of pBSK(); in this
case, the orientation of the insert was such that the NotI
site of pBSK() was located upstream of novH. A 5.5-kb
NotI fragment from this construct, which contained the ORFs
from novH to novK, was cloned into pBSK(),
providing a new XbaI site downstream of novK. The
insert of the resulting plasmid was excised with EcoRI and
XbaI and was cloned into the same sites of pUWL201 to give pMS75.
(v) pMS73. After deletion of novM and novN from p9-6GE9-R by PflMI and SpeI digestion, blunt ends were created with T4 DNA polymerase and the construct was religated. The insert of the resulting plasmid, which comprised the genes from novH to novL, was excised with EcoRI and XbaI and was ligated into the same sites of pUWL201 to give pMS73.
Transformation of expression plasmids into S. lividans TK24. Preparation of S. lividans TK24 protoplasts was carried out by the standard protocol (20) with cells grown for 48 h. Polyethylene glycol-induced protoplast transformation (20) was carried out with 10 µg of plasmid DNA per transformation. After protoplast regeneration on R2YE agar medium for 14 h at 28°C, transformants were selected by overlaying each R2YE plate with 200 µl of thiostrepton solution (5 mg/ml).
Preparation of cell-free extracts and novobiocic acid synthetase assay. For expression of novobiocic acid synthetase, S. lividans TK24 transformants were cultured in CDM medium as mentioned above. All further operations were carried out at 4°C. Cells were harvested and washed twice with 50 mM Tris-HCl (pH 8.0). Cells (3 g [fresh weight]) and 3 ml of buffer A (50 mM Tris-HCl [pH 8.0], 5 mM dithiothreitol, 50 µM phenylmethylsulfonyl fluoride) were ground for 20 min in a mortar to which sea sand had been added. The mixture was centrifuged for 30 min at 27,000 × g, and the supernatant was passed through a Sephadex G-25 column (Nap-10; Pharmacia, Uppsala, Sweden), which had been equilibrated with buffer A. Protein contents were determined by the method of Bradford (5).
Ring A was obtained by hydrolysis of novobiocin (30). Ring B and novobiocic acid were kindly provided by Pharmacia & Upjohn, Inc. (Kalamazoo, Mich.). The enzyme assay (100 µl) contained 100 µg of protein and the reagents described elsewhere (30), plus 5 mM MnCl2. The reaction was stopped after incubation for 20 min at 30°C by addition of 5 µl of 1.5 M trichloroacetic acid. After extraction with 1 ml of ethyl acetate, the organic phase was evaporated, and the residue was dissolved in H2O-methanol (50:50; vol/vol) and analyzed by high-pressure liquid chromatography (HPLC) as described in the section Analysis of metabolites.Construction of vector pAM1 for insertional gene inactivation. A 2.1-kb EcoRI fragment from cosmid 9-6G, which comprises part of the novT gene, was cloned into the EcoRI site of vector pBSKT, a pBluescript SK(+) derivative containing the thiostrepton resistance gene (36), resulting in pAM1. Restriction analysis showed that the novT gene fragment had the same orientation as the thiostrepton and the ampicillin resistance genes of the vector.
Transformation of S. spheroides NCIB 11891. Preparation of protoplasts was carried out as described elsewhere (22). Mycelia were grown for 48 h, harvested, and incubated with 10 mg of lysozyme in 5 ml of P buffer (20) for 60 to 90 min at 30°C.
For transformation, 10 to 50 µg of plasmid pAM1 was converted to the single-stranded form by incubation at 100°C for 10 min followed by rapid chilling on ice. The plasmid DNA was mixed with approximately 9.6 × 107 freshly prepared protoplasts (200 µl) under addition of 500 µl of T buffer (20) containing 50% (wt/vol) polyethylene glycol 1000 (Roth, Karlsruhe, Germany) and 30 µg of calf thymus DNA. Protoplasts were regenerated on R2YE agar medium overlaid with 3 ml of R3 agar (58), which was modified by inclusion of 0.8% (wt/vol) agar and 17.1% (wt/vol) sucrose and omission of disodium succinate and yeast extract. After 48 h at 25°C the plates were overlaid with 3 ml of soft nutrient agar (20) containing 0.33 mg of thiostrepton per ml for selection of integration mutants.Analysis of metabolites. Integration mutants of S. spheroides were cultured in CDM medium (see above) and were harvested by centrifugation. The medium (105 ml) was acidified with HCl to pH 2.0 and was extracted twice with 150 ml of ethyl acetate. The organic phase was washed twice with 100 ml of water and was dried with Na2SO4. The solvent was removed and the residue was dissolved in 1 ml of methanol.
Metabolites were analyzed by HPLC with a Multosphere RP18-5 column (250 by 4 mm; 5 µm; C+S Chromatographie Service, Düren, Germany) with a linear gradient from 60 to 100% methanol in 1% aqueous formic acid and detection at 305 nm. Authentic novobiocin (Fluka, Buchs, Germany) and novobiocic acid were used as standards. For preparative isolation, the fractions from HPLC analysis were collected and the solvent was evaporated. To remove residual formic acid, the residue was dissolved in 50 ml of water and the products were extracted with ethyl acetate. The organic layer was washed with water until the pH was neutral. After evaporation of the ethyl acetate, the product was analyzed by mass spectrometry (MS) and 1H-nuclear magnetic resonance (1H-NMR) spectroscopy. Electron impact mass spectra were recorded on a TSQ70 spectrometer (Finnigan, Bremen, Germany) by using methanol as the solvent. A molecular weight of 395.1 was observed (novobiocic acid is C22H21NO6; molecular weight, 395.4). 1H-NMR spectrum was measured on an AMX 400 spectrometer (Bruker, Karlsruhe, Germany), and the isolated compound gave signals corresponding to novobiocic acid:
ppm
(CD3OD) 7.75 (d, 2.25 Hz, H-2), 7.71 (dd, 8.52 Hz, 2.25 Hz,
H-6), 7.66 (d, J = 8.95 Hz, H-5'), 6.85 (d, 8.98 Hz,
H-6'), 6.84 (d, 8.53 Hz, H-5), 5.35 (t, sep 7.63 Hz, 1.5 Hz, H-8), 3.34 (H-7, overlapped with signals of solvent), 2.26 (s,
CH3-8'), 1.76 (s, H-11), 1.74 (s, H-10).
Nucleotide sequence accession number. The nucleotide sequence reported on here is deposited in the GenBank database under accession no. AF170880.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Screening of a cosmid library for the novobiocin biosynthetic gene cluster. Previous attempts to clone the novobiocin biosynthetic gene cluster by complementation experiments with novobiocin-deficient mutants of S. niveus were unsuccessful (9). Screening experiments with novobiocin resistance genes have been discussed as an alternative cloning strategy for this cluster (43, 52, 62), but no results have been reported so far. Therefore, we tried a different strategy. The sugar moiety of novobiocin (ring C) is a 6-deoxyhexose, and 6-deoxyhexoses are generally formed via a deoxynucleoside diphosphate (dNDP)-hexose 4,6-dehydratase reaction (Fig. 1, reaction II) (35, 46). Degenerate primers for the conserved N-terminal sequence of dNDP-glucose 4,6-dehydratase genes (10) were kindly provided by A. Bechthold, and PCR amplification of S. spheroides NCIB 11891 chromosomal DNA yielded two different fragments which could be identified as dNDP-glucose 4,6-dehydratases by sequence homology.
A cosmid library from S. spheroides was constructed in SuperCosI, and screening was carried out by using the two different dNDP-glucose 4,6-dehydratase gene fragments as probes. This led to the detection of two different groups of cosmids. Group 1 hybridized with a 2.1-kb EcoRI fragment; group 2 hybridized with a 6-kb EcoRI fragment. The cosmids were analyzed by conventional restriction mapping, as well as by hybridization of partial digests of cosmids to the SuperCosI vector sequences flanking the cosmid inserts (50). This showed that each group consisted of four different overlapping cosmids and contained only a single gene that hybridized with the respective probe. Random sequencing of fragments from both clusters revealed a sequence in cosmid group 1 with a very high degree of homology to gyrBr, which encodes a novobiocin-resistant gyrase B subunit and which has been described as the principal novobiocin resistance gene in S. spheroides (61). Hybridization experiments with this gene as the probe confirmed its presence in all four cosmids of group 1, but no homologous sequence was found in the cosmids of group 2 (data not shown). Since resistance genes are usually part of the biosynthetic gene clusters of the respective antibiotics, cosmid group 1 likely contained the biosynthetic genes for novobiocin and was further analyzed by sequencing.Sequence analysis of novobiocin gene cluster.
A total span of
53.1 kb was sequenced by single-strand sequencing, and a core sequence
of 25.6 kb was analyzed by double-strand sequencing. The core sequence
has been deposited in the GenBank database under accession no.
AF170880. The deduced gene organization of the identified 23 putative
ORFs within the core region is shown in Fig.
2A. Remarkably, 22 of the ORFs are
oriented in the same direction. Table 1
lists the ORFs identified and the homologies to database entries
identified.
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Gene insertional inactivation of the novobiocin biosynthetic gene
cluster.
In order to confirm that the cloned sequence indeed
represented the novobiocin biosynthetic gene cluster, an inactivation experiment was carried out with a 2.1-kb EcoRI fragment
containing parts of the dTDP-glucose 4,6-dehydratase gene
(novT) and of the putative C-methyltransferase
gene (novU). This fragment was cloned into pBSKT, which
carries a thiostrepton resistance gene, and the resulting plasmid was
introduced into S. spheroides protoplasts. Resistant clones
that arose from homologous recombination were isolated and analyzed by
Southern hybridization with a 520-bp FspI-PstI
probe derived from the dTDP-glucose 4,6-dehydratase gene; hybridization
bands appeared as expected at 3.98 and 1.47 kb after restriction with
AccI and at 1.07 and 0.91 kb after restriction with
PvuII, proving integration of the 2.1-kb EcoRI
fragment into chromosomal S. spheroides DNA by homologous
recombination (Fig. 4).
Thiostrepton-resistant transformants were tested for their secondary
product formation by HPLC analysis. In contrast to the wild type, no
novobiocin was detected, but another peak of similar UV absorption was
observed (Fig. 5). This substance was
isolated on a preparative scale. Both 1H-NMR and MS
analyses (see Materials and Methods) allowed the unequivocal
identification of this substance as novobiocic acid, i.e., the aglycone
moiety of novobiocin (Fig. 1).
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Heterologous expression of novobiocic acid synthetase in S. lividans TK24. A key reaction in the biosynthesis of novobiocin is the formation of the amide bond between ring A and ring B in an ATP-dependent reaction catalyzed by novobiocic acid synthetase (Fig. 1). An assay method for this enzyme, as well as the chemical synthesis of ring A and ring B from novobiocin, has been described previously (30). In our sequence analysis, NovH was shown to have homology to peptide synthetases and therefore was a likely candidate for the enzyme catalyzing the novobiocic acid synthetase reaction.
A 9.7-kb EcoRI fragment containing the genes from novH to novN was cloned into pEM4, a Streptomyces-E. coli shuttle vector containing the ermE up promoter for foreign gene expression, resulting in plasmid pSL3(+) (Fig. 2B). This construct was transformed into S. lividans TK24. Novobiocic acid synthetase activity could clearly be detected in cell extracts from that transformant but not in extracts from S. lividans transformed with an empty vector (Table 2).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The present study describes, for the first time, the cloning of a biosynthetic gene cluster of an aminocoumarin antibiotic. This class of antibiotics comprises novobiocin and two closely related compounds, coumermycin A1 and clorobiocin (41); the characteristic 3-amino-4,7-dihydroxycoumarin moiety is also found in a number of other antibiotics, such as protorubradirin (2).
In the novobiocin producer S. spheroides, we detected two different dNDP-glucose 4,6-dehydratase genes. Screening of a cosmid library with these two genes as probes revealed two distinct cosmid groups. The novobiocin resistance gene gyrBr was found in only one of the two cosmid groups, and the gyrB gene-containing group was shown to contain the novobiocin biosynthetic gene cluster. The other dNDP-glucose 4,6-dehydratase gene may therefore be involved in the biosynthesis of a metabolite different from novobiocin.
Sequence analysis of the novobiocin biosynthetic gene cluster revealed several genes for which possible roles in novobiocin biosynthesis could be putatively assigned by their homology to known genes in the database. Especially, an analogy to the biosynthesis of other deoxysugars and the results of earlier feeding experiments with novobiocin (3, 33) permit one to make a detailed hypothesis about the biosynthesis of the noviose moiety and the genes involved (Fig. 1). Glucose-1-phosphate is expected to be activated by nucleotidylation to give dTDP-glucose (Fig. 1, reaction I) and to be further converted to dTDP-4-keto-6-deoxyglucose under catalysis of an dNDP-glucose 4,6-dehydratase (reaction II). Subsequent steps include 3,5-epimerization and 4-ketoreduction (reactions III and IV). C-methylation at C-5" of the sugar (reaction V) may be the last of these five steps, but it could also occur at an earlier stage.
As shown in Table 1 and Fig. 2, four ORFs, grouped closely together immediately upstream of the gyrBr gene, could be fitted perfectly to the proposed noviose biosynthetic pathway: NovV, NovT, NovW, and NovS may catalyze reactions I to IV, respectively, of the reaction sequence shown in Fig. 1.
Both novU and novO show similarity to methyltransferases and represent candidates for the C-methylation reactions at C-5" of noviose (Fig. 1, reaction V) and at C-8' of ring B (Fig. 1, reaction X). NovU resembles EryBIII, which has been functionally shown to catalyze the C-methylation in the biosynthesis of dTDP-L-mycarose, the deoxysugar moiety of erythromycin, in Saccharopolyspora erythraea (17). This, as well as its association with the four other deoxyhexose biosynthetic genes, suggests that NovU rather than NovO catalyzes the C-5" methylation in noviose biosynthesis. The 4.8-kb fragment that comprises novS, novT, novU, novV, and novW therefore most likely contains the entire biosynthetic information for the biosynthesis of the deoxysugar noviose except 4-O-methylation, which takes place at a later stage.
C-4" O-methylation and C-3" O-carbamoylation (Fig. 1, reactions VIII and IX, respectively) are regarded as the last steps in novobiocin biosynthesis (31). Sequence data show two ORFs, novP and novN, which can putatively be assigned as O-methyltransferase and O-carbamoyltransferase, respectively.
Little information is available on the biosynthetic steps involved in the biosynthesis of ring A and ring B (Fig. 1). At present, an assignment of the genes detected in our study to reactions in the biosynthesis of rings A and B would be purely speculative. Formation of ring A includes a prenylation reaction, and the responsible dimethylallyl diphosphate:4-hydroxyphenylpyruvate dimethylallyl transferase has been identified in cell extracts of S. spheroides (60). Unfortunately, the presently known prenyltransferases that attach a prenyl group to an aromatic ring share very little homology. So far, none of the ORFs detected in our study can be suggested to be likely candidates for this enzyme.
Downstream of the gyrBr gene, several ORFs with homology to primary metabolic enzymes were detected, e.g., ORFs with homology to glyceraldehyde 3-phosphate dehydrogenase and to peptide and sugar transporters (data not shown). This suggests that the gyrBr gene may represent the right border of the cluster in Fig. 2. In contrast, the position of the border on the left end remains ambiguous, since, except for a subtilisin-like protease and two ATP-binding cassette transporters, no meaningful homologies were found in the region to the left of the core sequence depicted in Fig. 2 (data not shown).
Functional proof that the sequence that was identified contains the novobiocin gene cluster was provided by an insertional gene inactivation experiment. The resulting integration mutants contain a tandem duplication of novT and novU, including one intact and one truncated copy of each gene (Fig. 4). The integration led to a complete abolishment of novobiocin production, and the mutants accumulated the aglycone of novobiocin, i.e., novobiocic acid, instead. Since no promoter is apparently located between novT and novU (intergenic region, 54 bp), integration most likely disrupted the transcription unit that comprises the genes involved in deoxysugar formation.
Conflicting hypotheses have been made about the sequence of the linkage of the three rings, rings A, B, and C (Fig. 1), in the biosynthesis of novobiocin (19, 29). The fact that novobiocic acid (ring A and ring B) accumulated after disruption of deoxysugar (ring C) biosynthesis suggests that formation of the amide bond between ring A and ring B takes place prior to glycosylation. The gene product of novM is a likely candidate for the glycosyltransferase that connects ring C to novobiocic acid (Fig. 1, reaction VII).
Further proof for the function of the sequence was given by the heterologous expression of novobiocic acid synthetase. This enzyme catalyzes the linkage of ring A and ring B by an ATP-dependent amide bond formation (Fig. 1, reaction VI). Novobiocic acid synthetase activity could be demonstrated upon heterologous expression of a 9.7-kb fragment which included novH and six other ORFs of the cluster (Table 2). novH showed significant homologies to several peptide synthetases involved in the biosynthesis of biologically active peptides and contained eight conserved motifs expected in the adenylation domain of a single peptide synthetase module, as well as the 4'-phosphopantetheine attachment motif (Fig. 3). The general scheme of nonribosomal peptide bond formation (for reviews, see references 28 and 40) includes activation of each amino acid (or amino acid derivative) as an aminoacyl adenylate, followed by covalent thioester linkage with an enzyme-attached 4'-phosphopantetheine cofactor and transfer of the activated carboxyl group to the amino group of the next acyl intermediate; for each amino acid (or derivative) included in the peptide, these functions are assembled on a distinct peptide synthetase module. Amide bond formation between ring A and ring B of novobiocin by a similar mechanism may therefore require the existence of a second peptide synthetase module, and in fact, NovH alone was found to be unable to catalyze novobiocic acid formation (Table 2). Successive shortening of the 9.7-kb fragment (Table 2) showed that the inclusion of novL was essential for the enzymatic activity. novL is a further member of the superfamily of adenylate-forming enzymes (63). The deduced amino acid sequence exhibited ATP binding and ATPase motifs (Fig. 3) and showed homology to acyl-coenzyme A synthetases (51, 21), which are similar to peptide synthetases in their adenylation domain. Further enzymatic and genetic investigations are now in progress to elucidate the exact roles of novH and novL for the novobiocic acid synthetase reaction.
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ACKNOWLEDGMENTS |
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We thank D. A. Hopwood and E. Cundliffe for providing Streptomyces strains, A. Bechthold, J. A. Salas, and W. Piepersberg for donation of shuttle vectors pEM4, pBSKT, and pUWL201, and Pharmacia & Upjohn for supply of novobiocic acid and ring B. A. Bechthold, W. Wohlleben, and coworkers provided important technical advice for this project. We also thank Susanne Hennig and Emmanuel Wemakor for excellent technical assistance.
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (to L. Heide and S.-M. Li).
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FOOTNOTES |
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* Corresponding author. Mailing address: Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany. Phone: 49-7071-29-72460. Fax: 49-7071-29-5250. E-mail: heide{at}uni-tuebingen.de.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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