1 Vicuron Pharmaceuticals, via R. Lepetit 34, 21040 Gerenzano, Italy
2 Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, 9751 NN Haren, The Netherlands
Correspondence
Margherita Sosio
msosio{at}vicuron.it
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ABSTRACT |
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The GenBank accession number for the tcp gene sequence reported in this article is AJ605139.
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INTRODUCTION |
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In recent years, considerable progress has been made in understanding glycopeptide biosynthesis. Sequence information is now available for five gene clusters: chloroeremomycin (van Wageningen et al., 1998), balhimycin (Pelzer et al., 1999
; Recktenwald et al., 2002
), complestatin (Chiu et al., 2001
), A47934 (Pootoolal et al., 2002
) and A40926 (Sosio et al., 2003
). Characterization of several gene products overproduced in Escherichia coli and gene inactivation experiments in the producing strain (reviewed by Hubbard & Walsh, 2003
) have established the details of many biosynthetic steps. Most of this work has been carried out on the pathways of chloroeremomycin and balhimycin, two vancomycin-type glycopeptides.
Teicoplanin (Fig. 1, compound 1), produced by the actinomycete Actinoplanes teichomyceticus ATCC 31131, consists of a complex of closely related molecules differing in the nature of the acyl residue. A relevant structural feature of teicoplanin is the presence of an N-acetylglucosamine (GlcNAc) and an N-acylglucosamine residue attached to amino acids 6 and 4, respectively. Glycosyltransferases (GTFs) able to attach this type of sugar to glycopeptide aglycons have not been described yet (Solenberg et al., 1997
; Losey et al., 2001
, 2002
). With the objective of understanding the tailoring steps involved in decorating teicoplanin-type aglycons, we describe here the entire tcp cluster and report the overexpression of two active tcp GTF enzymes.
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METHODS |
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Expression of GTFs.
The genes encoding the two tcp GTFs (tGtfA and tGtfB) were amplified from cosmid DNA, using the following primer pairs: 5'-GCGCATATGCGCGTGCTGTTTTCGTCC-3' and 5'-GCAGATCTTCACGCGGGAACCGACGATC-3' for gtfA, 5'-GCGCATATGCGTGTGTTGTTGTCGACGAC-3' and 5'-CGCAGATCTCACGCGGAGACCGACGATCTCT-3' for gtfB. Forward and reverse primers introduced an NdeI and a BglII restriction site (underlined), respectively, into the PCR fragment. PCR reactions were carried out using Vent DNA polymerase (New England Biolabs) as described by the supplier. After NdeI and BglII digestion, the PCR fragments were cloned into the corresponding sites of the pET3B and pET15B expression vectors (Novagen), yielding the following constructs: ptGTFA, ptGTFB, pHis6tGTFA and pHis6tGTFB. All constructs were sequenced to ensure that no amplification errors had been introduced. These plasmids were introduced into E. coli BL21(DE3)-star (Stratagene). Subsequently, transformants were transferred to LB medium supplemented with 1 M sorbitol and incubated for 24 h at 30 °C. Cultures were centrifuged, washed and resuspended in 50 mM Tris/HCl buffer (pH 9). Cells were disrupted by two passages through a French pressure cell operated at 140 MPa. Crude extracts were prepared by centrifugation for 30 min at 40 000 g.
GTF activity assays and product analysis.
Enzymic activity was assayed in a total volume of 100 µl, containing 1 µmol UDP-GlcNAc and 100 nmol aglycon in 50 mM Tris/HCl buffer (pH 9) and a variable amount of cell extracts containing either or both tGtfA and tGtfB proteins. Reactions were incubated at 37 °C. After reaction termination on ice, products were identified with an analytical HPLC using an AlphaBond C18 column (250x4·6 mm, Alltech), applying a linear gradient from 1 % (v/v) acetonitrile in 0·1 % TFA to 33 % acetonitrile in 0·1 % TFA over 15 min at a 1 ml min-1 flow rate, with UV detection at 280 nm. Products formed in assays with combined extracts were analysed using a linear gradient from 1 % acetonitrile in 0·1 % TFA to 12 % acetonitrile in 0·1 % TFA over 15 min. New product peaks were analysed by HPLC-MS, by delivering 10 % of the post HPLC-column flow to a Navigator quadrupole mass spectrometer (Finnigan). Mass spectral analysis was performed in the positive-ion mode of an ESI mass spectrometer, operating over a range of 11501650 a.m.u. Nitrogen was used as nebulizing and drying gas. Ions were accelerated in a 25 V electric field. The teicoplanin aglycon and (pseudo)aglycon (compounds 2 and 4 in Fig. 1, respectively) have been described (Malabarba et al., 1984
) and were kindly provided by Sonia Maffioli (Vicuron Pharmaceuticals).
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RESULTS |
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Synthesis of HT probably involves the product of tcp ORF25, a putative non-haem iron dioxygenase (Table 1
). Homologues of this ORF are present in the dbv and sta clusters, but not in the bal and cep clusters. Thus, a common route for
HT formation seems to operate in teicoplanin, A47934 and A40926 biosyntheses, which differs from the balhimycin and chloroeremomycin case, where tyrosine is converted into
HT through the action of three enzymes (Chen & Walsh, 2001
; Puk et al., 2002
).
Synthesis of the heptapeptide precursor
Synthesis of the teicoplanin heptapeptide precursor is carried out by a non-ribosomal peptide synthetase (NRPS). These enzyme systems are organized in modules, with each module responsible for the recognition and incorporation of one amino acid into the oligopeptide (Marahiel, 1997). Within each module, the adenylation (A) domain is responsible for substrate recognition and activation, the thiolation (T) domain for forming a thioester bond with the cognate amino acid and elongating peptide, and the condensation (C) domain for catalysing peptide bond formation. In addition, an epimerization (E) domain is found in those modules epimerizing L-amino acids into their D-forms. The last module is usually completed by a thioesterase (Te) domain, which hydrolyses the thioester bond linking the completed peptide to the NRPS.
Four proteins, encoded by ORFs 9 to 12, constitute the seven-module teicoplanin NRPS. The genetic organization and domain composition of these ORFs indicate that ORF9, encoding the A-T-C-A-T-E domains, specifies modules 1 and 2; ORF10 (C-A-T) module 3; ORF11 (C-A-T-E-C-A-T-E-C-A-T) modules 4 to 6; and ORF12 (C-A-T-X*-Te, where X* denotes an atypical C or E domain of unknown function, so far present in all glycopeptide NRPSs) module 7. Thus, the tcp NRPS shows an overall domain composition identical to that of the bal, cep and dbv systems. Only the com and sta NRPSs present an E domain at the end of module 3, unexpected from the L-stereochemistry of the third amino acid of the heptapeptide in all glycopeptides.
Three other tcp ORFs are likely to participate in heptapeptide synthesis. ORFs 13 and 17 (Table 1) each encode a 69 aa polypeptide of unknown function, which is specified by an ORF located downstream of the NRPS gene encoding module 7 in the other glycopeptide clusters. The two polypeptides are completely identical over the first 60 aa, and present only 6 amino acid substitutions over their entire length. In addition, also the first 181 nt of the two ORFs are perfectly identical, suggesting that one copy arose from duplication of the other. The other ORF that may be indirectly involved in heptapeptide synthesis is ORF39, which encodes a type II thioesterase (Table 1
). The proposed role for these thioesterases is to hydrolyse misprimed or misacylated T domains (Heathcote et al., 2001
; Schwarzer et al., 2002
). A type II thioesterase-encoding gene is also present towards one end of the dbv cluster, but not in the bal, cep, com or sta clusters.
Cross-linking and halogenation of the aromatic residues
Teicoplanin contains three ether links (between amino acids 1-3, 2-4 and 4-6) and one CC link (between amino acids 5-7) joining its aryl groups. These cross-linking reactions are carried out by P450 mono-oxygenases (Bischoff et al., 2001a, b
), and the tcp cluster, like the sta and dbv clusters, contains four such oxy genes, designated ORFs 18, 19, 20 and 22 (Table 1
). It is therefore reasonable to assume that each Oxy protein carries a distinct cross-linking reaction. On the basis of the model proposed for the bal Oxy proteins (Bischoff et al., 2001b
) and using sequence similarity as the criterion, it is likely that ORF18 (77 % identical to bal-OxyA, but no more than 42 % identical to the other Oxy proteins) is involved in the cross-linking of the aromatic residues of amino acids 2 and 4; ORF20 (76 % identical to bal-OxyB, but no more than 50 % identical to the other Oxy proteins) in cross-linking of amino acids 4 and 6; and ORF22 (70 % identical to bal-OxyC, but no more than 47 % identical to the other Oxy proteins) for amino acids 5 and 7. By exclusion, ORF19 should be involved in cross-linking of amino acids 1-3.
Teicoplanin contains two chlorine atoms, one on the Tyr residue at position 2 and the other on the HT residue at position 6. The product of ORF21 (Table 1
) is highly related to other halogenases involved in chlorination of aromatic residues in glycopeptide biosynthesis (Puk et al., 2002
; Sosio et al., 2003
). Thus, a single halogenase should modify both residues.
Addition of sugars
Teicoplanin contains an N-acylglucosamine attached to the hydroxyl group of amino acid 4; a GlcNAc attached to the -hydroxy of amino acid 6; and a mannose residue attached to one of the hydroxyl groups of amino acid 7. The roles of the two NDP-dependent GTFs encoded by ORFs 8 and 23 (Table 1
) are described below. As proposed for the dbv cluster (Sosio et al., 2003
), mannosylation is likely to be carried out by a different type of GTF, unrelated to the tGtfA and tGtfB proteins described below. The putative mannosyltransferase encoded by the tcp cluster is specified by ORF15 (Table 1
).
Resistance, export, regulation and unknown functions
The above analysis accounts for 23 of the 39 tcp genes. Seven of the remaining 16 ORFs are grouped at the left end of the tcp cluster, where the vanHAX-like glycopeptide-resistance cassette (ORFs 2 to 4) is located next to a murF homologue (Sosio et al., 2000). Thus, the sta and tcp cluster share the same set of four genes at one end of the cluster, although they are differently organized. At least in the A47934 producer Streptomyces toyocaensis, these genes are important in conferring glycopeptide resistance (Pootoolal et al., 2002
). In vancomycin-resistant enterococci, homologues of ORFs 2 to 4 are involved in the synthesis of the D-alanyl-D-lactate moiety at the termini of the pentapeptide chains in nascent peptidoglycan, thus reducing the extent of glycopeptide binding to its molecular target (Evers et al., 1996
). ORFs 2 to 4 are therefore likely to be involved in conferring some level of teicoplanin resistance to the producing strain. It remains to be established whether ORF1, specifying a putative D-alanyl-D-alanine-adding enzyme, plays any role in conferring glycopeptide resistance.
ORFs 16 and 34 are likely to specify export functions, since they are predicted to encode an ABC-type and an ion-dependent transmembrane transporter, respectively (Table 1). ORFs 6 and 7 encode the response regulator and the sensor kinase, respectively, of a likely two-component signal transduction system (Table 1
). The product of ORF28 belongs to the family of StrR-type positive transcriptional regulators, while ORF29 specifies a positive regulator of the LuxR family (Table 1
).
ORFs 5, 14, 24, 26, 27 and 35 complete the list of tcp genes (Table 1). ORF5 is related to a protein of unknown function from Amycolatopsis orientalis, which however lies outside the cep cluster. ORFs 14 and 24 find matches in other glycopeptide clusters: the former is specified by the bal, cep and dbv clusters, while the latter only by the dbv cluster. ORF26 encodes a putative acyl-CoA ligase, while ORF27 encodes a putative 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of the AroA type, unrelated to the equivalent polypeptide encoded by the cep cluster. Finally, ORF35 shows considerable sequence identity to type I GTP cyclohydrolases.
Expression of GTFs
The gene composition of the tcp cluster makes it very likely that this region is indeed involved in the synthesis of a glycopeptide antibiotic. Since no methods for the genetic manipulation of A. teichomyceticus have been described, we sought to provide direct evidence for the function of selected tcp genes through heterologous expression. Of the two NDP-dependent GTFs identified in the tcp cluster, ORF8 is most related to GtfA proteins, typically glycosylating amino acid 6, and ORF23 to GtfBs, which specifically glycosylate amino acid 4. In order to establish their roles, both genes were cloned in the expression vectors pET3b and pET15B (the latter introducing an N-terminal His6 tag). SDS-PAGE analysis of centrifuged E. coli extracts revealed that both proteins, without the N-terminal His-tag, were expressed in E. coli cultivated in LB at 37 °C, but large amounts of insoluble protein were present in the pellet fractions. Also the His-tagged version of tGtfB yielded high amounts of insoluble protein, while no His6-tGtfA expression was observed in E. coli. Addition of 1 M sorbitol to the growth medium and decreasing the cultivation temperature from 37 to 30 °C resulted in a clear shift from insoluble to soluble proteins for tGtfA, tGtfB and His6-tGtfB (data not shown). However, the altered cultivation conditions did not lead to detectable His6-tGtfA protein. The use of compatible solutes, such as sorbitol, to stimulate correct protein folding and counteract inclusion body formation, has been described previously (Barth et al., 2000).
Incubations of E. coli extracts expressing tGtfA with teicoplanin aglycon (Fig. 1, compound 2) and UDP-GlcNAc resulted in the appearance of a new HPLC product peak (Fig. 3
, trace C). No product was observed in incubations of extracts derived from E. coli-pET3b cells (Fig. 3
, trace A). The retention time of the tGtfA-dependent peak coincided with that of authentic compound 4 (Fig. 3
, trace E). Consistently, HPLC-MS experiments indicated that the molecular mass of the tGtfA product peak was 1400·3, confirming that tGtfA transforms compound 2 into compound 4 (Fig. 1
). Incubation of the E. coli cell extracts expressing tGtfB or His6-tGtfB with teicoplanin aglycon and UDP-GlcNAc revealed the appearance of a novel peak (Fig. 3
, trace B), with a different retention time from compound 4 but with identical molecular mass. This result suggests that tGtfB adds a GlcNAc moiety to a different position in the teicoplanin aglycon, most likely yielding compound 3 in Fig. 1
. To determine whether the combined action of tGtfA and tGtfB produces a double glycosylated aglycon, crude extracts of both proteins were combined in one assay. HPLC analysis showed that this incubation yielded three product peaks, two of which corresponded to the products observed previously in single tGtf-incubations. The retention time of the novel product peak was very close to that of compound 3 (Fig. 3
, trace D). To achieve a reasonable separation of both compounds, a shallow gradient was applied, which yielded retention times of 9·2 and 8·7 min for compound 3 and the third product peak, respectively (Fig. 4
). MS analysis of the 8·7 min product peak yielded a molecular mass of 1603·4, as expected from compound 5 (Fig. 1
). The abundance of this peak increased with incubation time (Fig. 4
).
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When cell extracts containing either tGtfA or tGtfB were incubated with vancomycin aglycon and UDP-GlcNAc, no HPLC product peak was observed, not even after 24 h incubation. This is in contrast with the cep-encoded GtfB enzyme, which also glycosylates the teicoplanin aglycone at amino acid 4 (Losey et al., 2001, 2002
). UDP-sugar substrate specificity was tested for both tGtf enzymes in incubations with teicoplanin aglycon. The activated sugars tested were: UDP-N-acetylgalactosamine, U/TDP-glucose and UDP-galactose. No conversion of teicoplanin aglycon was found with any of these activated sugars.
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DISCUSSION |
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One unique feature of the tcp cluster is the lack of an ORF encoding a putative prephenate dehydrogenase (PDH). PDH-encoding ORFs have been observed so far in the five other glycopeptide clusters, as well as in the cda (Hojati et al., 2002) and ramoplanin (Zazopoulos et al., 2002
) clusters from Streptomyces coelicolor and Actinoplanes sp. ATCC 33076, respectively. The corresponding compounds contain an HPG residue. The presumed role of PDH is to prime HPG synthesis by generating p-hydroxyphenylpyruvate (HPP), the first substrate for the enzyme Hmo. It should be noted that, using Tyr as an amino donor, HpgT generates one molecule of HPP. In the actinomycetes where this has been investigated, Tyr biosynthesis requires an arogenase, which converts prephenate into arogenate (Hodgson, 2000
). In addition, the S. coelicolor genome (Bentley et al., 2002
) contains a single PDH-encoding gene within the cda cluster. We do not know whether A. teichomyceticus possesses a PDH-encoding gene somewhere else in its genome. However, it should be noted that, in those glycopeptides where both HPG and DPG are utilized, HPP can be formed by transamination of Tyr during DPG formation. Thus, a PDH activity may be strictly necessary only in those cases when HPG synthesis is unlinked to that of DPG.
The gene(s) responsible for N-acylation of the glucosamine residue have not been identified. Contrary to our expectations, the tcp cluster specifies no homologue of dbv ORF23, which contains motifs typical of the family 3 of acyltransferases (Sosio et al., 2003) and could have been a likely candidate for carrying out this reaction. Instead, the tcp cluster specifies a putative acyl-CoA ligase, encoded by ORF26 (Table 1
). If N-acylation is carried out by homologous genes during teicoplanin and A40926 biosynthesis, then the dbv and tcp clusters should share a gene absent from the other glycopeptide clusters. The only ORFs of unassigned function fitting this requirement are represented by tcp ORF24 and dbv ORF8. However, these sequences show no homology in database searches. An alternative possibility is that N-acylation is carried out through different mechanisms in A40926 and teicoplanin formation. The existence of two different pathways for
HT formation represents a precedent in glycopeptides.
We observed that compound 5 is formed by the combined action of tGtfA and tGtfB while compound 4 is not a substrate for tGtfB. This suggests that tGtfA can glycosylate the teicoplanin molecule independently of the glycosylation at amino acid 4, while tGtfB can act only on the aglycon. This would imply that tGtfB acts before tGtfA during teicoplanin biosynthesis in A. teichomyceticus. In vivo studies with the balhimycin producer indicated that a bgtfB deletion mutant produced only the balhimycin aglycon (Pelzer et al., 1999), suggesting that bGtfA is unable to attach epi-vancosamine to amino acid 6. If this is true also in vitro, it would mean that bGtfA, unlike tGtfA, can only recognize a balhimycin pseudoaglycon glucosylated at amino acid 4.
A second finding is that tGtfB recognizes UDP-GlcNAc as the substrate. Since we did not have access to UDP-glucosamine, we do not know whether this reflects a relaxed specificity by tGtfB. The alternative possibility is that UDP-GlcNAc is the true substrate for tGtfB, and then a transacylation reaction replaces the acetyl group on this sugar with the fatty acid chain found in teicoplanin. This latter hypothesis is also consistent with the utilization of UDP-GlcNAc by several cellular pathways, while we are unaware of instances where bacterial metabolism uses an NDP-activated glucosamine.
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ACKNOWLEDGEMENTS |
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Received 22 May 2003;
revised 12 September 2003;
accepted 3 October 2003.
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