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• W2149776410 abstract "Teicoplanin biosynthesis. In vitro studies have revealed that the clinically important N-acyl-glucosaminyl residue on teicoplanin originates from UDP-N-acetyl-glucosamine. A deacetylase essential for this biosynthetic pathway has been identified and characterised, and might prove useful in the production of novel glycopeptides. The glycopeptide antibiotics are an important class of natural products biosynthesised by the actinomycetes. The prevalence of methicillin-resistant Staphylococcus aureus (MRSA)1 means that vancomycin and teicoplanin are crucial antibacterial agents, but the emergence of glycopeptide-resistant bacteria such as vancomycin-resistant Enterococcus (VRE)2 and Staphylococcus aureus (VRSA)3 necessitates the development of new antibiotics. The chemical modification of natural glycopeptide scaffolds is proving a successful route to clinically useful compounds,4 although a potentially more attractive route to novel glycopeptides is through engineered biosynthesis. Teicoplanin (Scheme 1) is a lipoglycopeptide produced by Actinoplanes teichomyceticus5 whose aglycone is decorated with N-acyl-D-glucosamine, N-acetyl-D-glucosamine and D-mannose at amino acids 4, 6 and 7, respectively. It has been demonstrated that these sugars and the N-acyl group are important for antibacterial efficacy,6 so an understanding of the biosynthesis of these moieties is desirable for the rational engineering of glycopeptide biosynthesis. The biosynthesis of the N-acyl-glucosamine group is of particular importance, as lipidated glycopeptides anchor themselves to the bacterial cell membrane,7 whereas nonlipidated homologues are more broadly distributed. It has been proposed that the lipid chain increases the local concentration of the antibiotic near the peptidoglycan, the antibacterial target for glycopeptides. Additionally, Dong et al.8 attributed teicoplanin's activity against the VRE VanB phenotype to its hydrophobic chain. Structures of teicoplanins. The amino acids are numbered in grey. The biosynthetic gene cluster for teicoplanin was elucidated by our research group9 and independently by Donadio et al.10 It was established that Orf10* is the glycosyltransferase responsible for sugar attachment to amino acid 4, and Orf11* is an acyltransferase that attaches a fatty acyl chain to glucosaminyl pseudoaglycone 3.9, 11 Orf10* is a member of the family 1/family 28 NDP–sugar glycosyltransferase superfamily (NDP=nucleotide diphosphate),12 so it requires an NDP–sugar as the sugar donor. It has not been determined whether this NDP–sugar is a product of primary or secondary metabolism in A. teichomyceticus;9 examples of both have been characterised in other biosynthetic pathways. The teicoplanin gene cluster contains an open-reading frame (ORF), orf2*, that encodes a homologue to BtrD, a nucleotidyltransferase in the butirosin gene cluster that catalyses TDP–glucosamine formation from TTP and glucosamine-1-phosphate.13 It is proposed that this activated sugar is then transferred to 2-deoxystreptamine by a putative glycosyltransferase, BtrM. Butirosin biosynthesis therefore appears to represent an example in which the NDP–sugar is a product of secondary metabolism. An important alternative pathway is represented by N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside deacetylase (MshB), a zinc-dependent deacetylase in mycothiol biosynthesis.14 Here, the NDP–sugar is a primary metabolite, UDP-N-acetyl-glucosamine (UDP-GlcNAc). After glycosyltransfer of GlcNAc to myo-inisitol, MshB catalyses the hydrolytic removal of the acetyl group. Homologous pathways exist in the biosynthesis of glycosylphosphatidylinositol15 (deacetylation carried out by PIG-L) and Lipid A16 (LpxC). Intriguingly, Orf2* also contains significant sequence similarity to MshB and PIG-L. Therefore, it is likely that Orf2* functions as either a nucleotidyltransferase or a deacetylase (Scheme 2). Putative pathways for the biosynthesis of the N-acyl-glucosamine moiety. In this communication, we wish to report that Orf2* is a deacetylase and is not a nucleotidyltransferase, and as such catalyses a crucial step in the maturation of teicoplanin. We also demonstrate that this enzyme might prove useful in the semisynthetic production of novel glycopeptides. The phylogenetic implications of this result are discussed. The orf2* gene was cloned into the pET28a(+) expression vector, and the protein was expressed in E. coli Rosetta(DE3) as an N-terminal His6-tagged protein. This was purified by nickel-affinity chromatography and size-exclusion chromatography. The His6 tag was cleaved by thrombin digestion, and the mass was determined to be 30 344 kDa by LC/ESI-MS. This is in excellent agreement with the predicted mass of 30 349 kDa (Orf2*+GlySerHis). The cloning and expression of Orf10* have been reported previously.9 Orf10* does not accept UDP-N-octanoyl-D-glucosamine as a substrate, so it is likely that the native substrate for Orf10* is UDP-N-acetyl-D-glucosamine or UDP-D-glucosamine (UDP-GlcN). These are both active substrates for Orf10*.9 Bacteria lack the machinery to produce UDP-GlcN, so a nucleotidyltransferase encoded within the gene cluster would be required for this pathway (Scheme 2). UDP-GlcNAc is a primary metabolite essential for the biosynthesis of the bacterial peptidoglycan, but an enzyme within the gene cluster would be required for deacetylation prior to acyltransfer. Sequence homology suggests that Orf2* could participate in either of these hypothesised pathways. The substrate specificity of Orf10* was analysed by a competitive assay, which is a simple method to compare the relative kcat/Km of two substrates. Orf10* was incubated with equimolar amounts of UDP-GlcNAc, UDP-GlcN and teicoplanin aglycone for 24 h and analysed by LC/ESI-MS. The relative sugar attachment for UDP-GlcNAc/UDP-GlcN was 5.9:1.0. The relative rate of transfer of N-acetyl-glucosamine and glucosamine to the aglycone provides strong evidence that UDP-GlcNAc is the native substrate. No sugars were transferred to the pseudoaglycones A3-1 and A3-2 (Scheme 3); this demonstrates that fully deglycosylated teicoplanin is the native sugar acceptor. Therefore, amino acid 4 is glycosylated first, followed by amino acid 6. Exogenous de-mannosylated teicoplanin is efficiently mannosylated by A. teichomyceticus cultures;17 this suggests that mannosylation is a late-stage event and is thus the final glycosylation step. Structures of teicoplanin A3-1 and A3-2. Relative rates of reaction might not provide a true picture of in vivo reactivity, as a nucleotidyltransferase producing NDP–glucosamine might boost the local concentration to sufficiently overcome this kinetic disadvantage. However, although it might be difficult to accurately determine in vivo NDP–sugar relative concentrations, this result implies that some transfer of N-acetyl-glucosamine to the aglycone would be expected (owing to the high natural abundance of UDP-GlcNAc) even if it is not the intended natural substrate. Since no N-acetyl-teicoplanin has been isolated, deacetylation is a likely biosynthetic step as Orf11* does not function as a transamidase.9 It was expected that the elucidation of Orf2*'s function would reveal the true biosynthetic pathway, due to its similarity to the N-acetyl-glucosamine deacetylases MshB and PIG-L, and the nucleotidyltransferase BtrD. Nucleotidyltransferase activity was analysed by incubation of the enzyme with NTP (UTP or TTP) and sugar-1-phosphate (GlcN-1-P, GlcNAc-1-P or Mannose-1-P). Analysis by ion-pairing HPLC18 revealed low-level activity with UTP and GlcNAc-1-P, but this was attributed to a trace amount of a coeluting nucleotidyltransferase, as activity diminished and was completely abolished as the enzyme was further purified. No deacetylase activity was detected with GlcNAc-1-P or UDP-GlcNAc, so the more likely pathway of deacetylation following glycosyltransfer was tested in a coupled assay with Orf2*, Orf10*, UDP-GlcNAc and teicoplanin aglycone (Figure 1 B). A control assay without Orf2* is shown for comparison (Figure 1 A). 1The appearance of a peak at 14.6 min with m/z 1359.3 is consistent with the formation of glucosaminyl pseudoaglycone 3. This retention time and m/z pattern is identical to a standard produced by using Orf10* and UDP-GlcN. Activity was tested in an isolated system by enzymatically synthesising N-Ac-glucosaminyl pseudoaglycone (2) by using Orf10*. Orf2* rapidly deacetylated 2, thus proving that the reaction does not cooperatively require Orf10*. These results demonstrate that Orf2* is a deacetylase and verify that UDP-GlcNAc is the true substrate for Orf10*. Deacetylation is much more rapid than glycosyltransfer, thus no acetylated intermediate is detected in the coupled assay and none is isolated from A. teichomyceticus. The biosynthesis of teicoplanin therefore represents another example where a glucosamine residue on a natural product originates from UDP-N-acetyl-D-glucosamine. Orf2* deacetylation monitored by LC/ESI-MS. A) Teicoplanin aglycone with UDP-GlcNAc and Orf10*; B) teicoplanin aglycone with UDP-GlcNAc, Orf10* and Orf2*. The basis for deacetylation activity can be rationalised by sequence analysis,19, 20 of Orf2* with some important homologues (Figure 2), including the known deacetylases MshB and human PIG-L, and TT1542, a protein of unknown function whose crystal structure has been determined.21 The cocrystallisation of MshB,22, 23 with Zn2+ and β-octylglucoside provides a detailed model of its active-site binding and catalysis. The alignment shows that almost all the residues proposed for MshB substrate binding and catalysis are present in all homologues, despite low overall sequence identity. In particular, there are two regions essential for catalytic activity that are conserved throughout all homologues: residues 15–19, (A/P)H(P/L/A)DD, and 161–164, HXD(H/N). In addition, the guanidinium group of Arg68 of MshB is proposed23 to hydrogen bond to the GlcNAc moiety, and this residue is conserved throughout the sequences analysed. An active-site model for Orf2* can therefore be constructed (Scheme 4). The identity of the proposed metal cofactor is as yet unknown. The addition of 10 mM EDTA had a negligible effect on enzyme activity; this suggests the presence of a very tightly bound metal cofactor or, perhaps, none at all. The addition of a range of divalent metals had little effect on activity, with the notable exception of Zn2+, which fully inhibited the enzyme despite being the proposed cofactor for MshB. Zinc inhibition is a common phenomenon among zinc-dependent enzymes due to inhibitory zinc(II) coordination to active-site residues.24 Further studies will be necessary to identify the native cofactor. Multiple alignment,19, 20 of Orf2* with seven homologues. Dbv21: Nonomuraea sp. ATCC 39727 (A40926 producer); Cep15: Amycolatopsis orientalis (chloroeremomycin producer); Orf2: Amycolatopsis balhimycina (balhimycin producer); MitC: Streptomyces lavendulae (mitomycin C producer); PIG-L: Homo sapiens (glycosylphosphatidylinositol biosynthesis); MshB: Mycobacterium tuberculosis (mycothiol biosynthesis); TT1542: Thermus thermophilus (unknown function). Fully conserved residues have white text on a red background, and similar (at least 70 %) amino acids are framed in blue (the similar residues are coloured red). Active-site model of Orf2* based on sequence homology to MshB. Asp18 functions as a general base, His16, Asp19 and His164 are ligands for a putative M2+ cofactor, leaving vacant coordination sites for water and the N-acetyl carbonyl oxygen. His161 might also stabilise the tetrahedral intermediate. The alignment data clearly show that Orf2 from the balhimycin gene cluster and Cep15 from the chloroeremomycin gene cluster contain significant sequence identity to Orf2* (65 % and 64 % identity to Orf2*, respectively), including all the residues implicated in deacetylation catalysis and substrate binding. However, these vancomycin-like glycopeptides do not contain any N-acetyl-glucosamine-derived moieties, neither does their proposed biosynthesis25 require a deacetylase. There are four plausible explanations: 1) their biosynthesis has not been correctly characterised; 2) these proteins have evolved an alternative function; 3) these genes are inactive evolutionary relics; 4) they are involved in the regulation or export of the compound. To determine which of these is true will require further work. Gene inactivation and in vitro characterisation of Cep15 is currently in process. It is worth noting that Orf2 and Cep15 possess an asparagine in place of a histidine as the third putative M2+ ligand (Figure 2). 2This is unique amongst homologues and might explain an alternative or null function. The teicoplanin-like lipoglycopeptides A40926 and aridicin can be deacylated by incubation with A. teichomyceticus ATCC 31121.26, 27 This process is a synthetically useful biotransformation as the resultant free amine can be derivatised (chemically or enzymatically) to yield novel glycopeptides.27 This is the same amide bond that Orf2* hydrolyses in teicoplanin biosynthesis, so Orf2*'s ability to deacylate teicoplanin was examined (Figure 3). This reaction produced a more hydrophilic compound at 13.0 min with m/z 1724.2. This m/z pattern is consistent with deacylteicoplanin (5; predicted [M+H]+=1724.4), and the transformation is rapid enough to be synthetically useful (0.16 μmol h−1 per mg protein). The peak at 17.4 min is teicoplanin A3-1 (Scheme 3), a factor that lacks the N-acyl-glucosamine residue at amino acid 4. It is unaffected by Orf2*, thus demonstrating the regioselectivity of this biotransformation. To assess why A. teichomyceticus does not produce any deacylated teicoplanin, a competitive assay was performed between equimolar amounts of teicoplanin and 2. Full deacetylation of 2 occurred with only a 6 % conversion of teicoplanin to 5, thus demonstrating that teicoplanin's kcat/Km is at least 20 times smaller than 2's kcat/Km. In addition to being much less reactive than the native substrate, reacylation of 5 by Orf11* coupled to the efficient export of teicoplanin by Orf4*9 will minimise the in vivo production of 5. Deacylation of teicoplanin monitored by LC/ESI-MS. A) Natural teicoplanin LC trace; B) teicoplanin after incubation with Orf2* for 16 h. In summary, we have identified a deacetylase that is involved in a crucial step in the biosynthesis of the N-acyl-glucosaminyl moiety of teicoplanin. In doing so, we have confirmed that the natural substrate for the glycosyltransferase Orf10* is UDP-GlcNAc. This activity is rationalised by comparison with homologous enzymes. Orf2* is also capable of deacylating teicoplanin, a regiospecific biotransformation that might be synthetically useful. It is highly possible that homologous pathways exist in the biosynthesis of other glucosamine-containing natural products whose gene clusters contain Orf2* homologues. Such natural products include A40926,29 a number of aminoglycosides13 and mitomycin, for which feeding studies demonstrated that its mitosane core is partially derived from (a probably activated) D-glucosamine.30 J.B.S. wishes to thank the BBSRC for financial support. A.W.T. would like to thank the BBSRC and St. John's College Cambridge for a studentship. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2268/2006/z600308_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article." @default.
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• W2149776410 date "2006-10-26" @default.
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• W2149776410 title "Identification of a Deacetylase Involved in the Maturation of Teicoplanin" @default.
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• W2149776410 doi "https://doi.org/10.1002/cbic.200600308" @default.
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