CHEMBIOCHEM FULL PAPERS DOI: 10.1002/cbic.201402179

Harnessing the Synthetic Capabilities of Glycopeptide Antibiotic Tailoring Enzymes: Characterization of the UK-68,597 Biosynthetic Cluster Grace Yim, Lindsay Kalan, Kalinka Koteva, Maulik N. Thaker, Nicholas Waglechner, Irene Tang, and Gerard D. Wright*[a] In this study, a draft genome sequence of Actinoplanes sp. ATCC 53533 was assembled, and an 81-kb biosynthetic cluster for the unusual sulfated glycopeptide UK-68,597 was identified. Glycopeptide antibiotics are important in the treatment of infections caused by Gram-positive bacteria. Glycopeptides contain heptapeptide backbones that are modified by many tailoring enzymes, including glycosyltransferases, sulfotransferases, methyltransferases, and halogenases, generating extensive chemical and functional diversity. Several tailoring enzymes in

the cluster were examined in vitro for their ability to modify glycopeptides, resulting in the synthesis of novel molecules. Tailoring enzymes were also expressed in the producer of the glycopeptide aglycone A47934, generating additional chemical diversity. This work characterizes the biosynthetic program of UK-68,597 and demonstrates the capacity to expand glycopeptide chemical diversity by harnessing the unique chemistry of tailoring enzymes.

Introduction Glycopeptide antibiotics (GPAs), such as the natural products vancomycin, teicoplanin, and the semi-synthetic telavancin, are important for the treatment of Gram-positive infections, particularly those caused by staphylococci or enterococci. Unfortunately, the emergence of antibiotic resistance is making the treatment of infections caused by Gram-positive bacterial pathogens even more challenging.[1] Some of the most problematic resistant bacterial strains include vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant S. aureus (VISA, hVISA, VRSA). It is essential that new antibiotics with the potential to treat VRE and other Gram-positive infections continue to be developed. Several semi-synthetic GPAs are currently in the drug pipeline of development and approval. For example, dalbavancin is an analogue of the GPA A40926 with a 3,3-dimethylaminopropyl amide substitution on the peptide carboxyl group[2] that recently received FDA approval. Oritavancin, a semi-synthetic derivative of chloroeremomycin with a 4’-chlorobiphenylmethyl substituent on the disaccharide sugar,[3] is undergoing priority review by the FDA. Telavancin, a derivative of van-

[a] G. Yim,+ L. Kalan,+ K. Koteva, M. N. Thaker, N. Waglechner, I. Tang, Dr. G. D. Wright Department of Biochemistry and Biomedical Sciences The M. G. DeGroote Institute for Infectious Disease Research McMaster University Hamilton, ON, L8N 3Z5 (Canada) E-mail: [email protected] [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201402179.

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comycin with a hydrophobic tail added to the vancosamine sugar, was recently approved for clinical use in Canada and the United States.[4] These semisynthetic lipoGPAs show improved efficacy against both vancomycin-sensitive and -resistant organisms while imparting desired pharmacological properties, such as increased systemic half-life in comparison to vancomycin, illustrating that judicious chemical scaffold modification can improve clinically relevant antibiotics.[2, 5] There are numerous members in the GPA class of antibiotics, all sharing a common topography, including a heptapeptide backbone that is crosslinked by oxygenases acting on aromatic amino acids to yield a cup-shaped scaffold. Two main peptide scaffolds exist, the vancomycin and teicoplanin types, which differ by the N-terminal three amino acids of the heptapeptide. Further diversification lies in the degree and specificity of modification by tailoring enzymes including mono-oxygenases, glycosyltransferases, halogenases, methyltransferases, acyltransferases, and sulfotransferases. GPA tailoring by these enzymes is one strategy to expand chemical diversity and to improve activity of existing GPAs via synthetic biology approaches.[6] Several groups have focused on the effects of glycosylation and the potentially promiscuous nature of the glycosyltransferases to accept different NDP-sugar substrates, chemically[7–9] and enzymatically producing novel glycosylated vancomycin[8–10] and teicoplanin[11] derivatives. Enzymes cloned from environmental DNA libraries have led to the synthesis of novel sulfated teicoplanin aglycone derivatives.[12, 13] Furthermore, GPA sulfation could prove to be especially important as sulfation of A47934, a teicoplanin-type antibiotic, blocks the activity of the sensor kinase VanS and attenuates the induction of GPA resistance.[14] The modification of GPA scaffolds by tailoring enChemBioChem 2014, 15, 2613 – 2623

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CHEMBIOCHEM FULL PAPERS zymes, therefore, has the potential to confer a wide variety of favorable characteristics on these antibiotics. With the costs of genome sequencing dramatically decreasing and new GPA biosynthetic gene cluster sequences becoming increasingly available,[15] the repertoire of tailoring enzymes imparting previously uncharacterized modifications or substrate specificity is also expanding.[6] Understanding the biosynthesis of this important class of antibiotics and harnessing the tailoring enzymes contained within can expand the chemical diversity of GPA antibiotics. Actinoplanes sp. ATCC 53533 is reported to produce the GPA UK-68,597[16] with a unique set of such tailoring enzymes that sulfate the dihydroxyphenylglycine at amino acid position 3 and chlorinate the p-hydroxyphenylpyruvate in the N-terminal amino acid position. Here, we describe sequencing and characterization of the UK-68,597 GPA biosynthetic cluster, the mining for unique tailoring enzymes, and their use in creating GPAs with novel chemistry.

Results UK-68,597 is in an unusual sulfated teicoplanin-type GPA with an N-terminal a-keto acid. It is produced by Actinoplanes sp. ATCC strain 53533, an actinomycete with yellow-orange substrate mycelium that does not produce aerial mycelium (Figure 1). This family of bacteria typically produces sporangiumcontaining motile spores.[17] The sequence of the UK-68,597 heptapeptide backbone, starting from the N terminus, is as follows: m-chloro-p-hydroxyphenylpyruvate-Tyr-Dpg-Hpg-HpgBht-Dpg (Hpg, 4-hydroxyphenylglycine; Tyr, tyrosine; Dpg, 3,5dihydroxyphenylglycine; Bht, b-hydroxytyrosine).[16] The heptapeptide has several interesting features, including an aromatic sulfate ester on the Dpg at amino acid 3 (Dpg3), four aromatic chlorinations, and an a-keto acid in place of the usual N-terminal amino acid (Figure 2). In our hands, this type strain did not

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Figure 1. Morphology of Actinoplanes sp. ATCC 53533 colonies grown on A) SAM or B) Bennett’s agar plates.

produce antibiotic, making the study of UK-68,597 challenging. We elected to explore its biosynthetic gene cluster as a prelude to transferring the genes encoding its tailoring enzymes to a more genetically amenable host in which GPA production has the potential to be enhanced.

The biosynthetic cluster of the GPA UK-68,597 A 454 pyrosequencing approach was used to obtain a draft sequence of the Actinoplanes sp. ATCC 53533 genome. The draft genome sequence contained 13.2 Mb of consensus sequence in 714 contigs (N50 59320 bp). Analysis of the genome with the natural product prediction software antiSMASH[18] projected the presence of 10 NRPS, PKS, and NRPS-PKS hybrid biosynthetic clusters. The Actinoplanes genome also had the predicted potential to produce indole, bacteriocin, siderophore, phosphonate, lantipeptide, and terpene molecules. One cluster was putatively related to glycopeptide biosynthesis, encoding an NRPS for a GPA-like heptapeptide scaffold and a type III PKS

Figure 2. Structure of UK-68,597 and a schematic of its biosynthetic cluster. See Table 1 for annotation. The letters S, Y, R, and H indicate the corresponding vanS, vanY, vanR, and vanH self-resistance genes. Numerals on the biosynthetic cluster indicate the auk numbering shown in Table 1.

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similar to DpgA, the first enzyme involved in the biosynthesis of amino acid Dpg. The putative GPA biosynthetic cluster was identified on a ~ 350 kb contig. Each open reading frame was identified and annotated, yielding an 81 kb span of DNA (Figure 2, Table 1, GenBank accession number KF192710). AntiSMASH[18] analysis of the nucleotide sequence encoding the heptapeptide cluster predicted a heptapeptide sequence

Table 1. Predicted open reading frames of the UK-68,597 biosynthetic cluster. Protein Putative annotation Ddlauk VanS VanY Auk1 Auk2 VanR VanH Auk3 Auk4 Auk5 Auk6 Auk7 Auk8 Auk9 Auk10 Auk11 Auk12 Auk13 Auk14 Auk15 Auk16 Auk17 Auk18 Auk19 Auk20 Auk21 Auk22 Auk23 Auk24 Auk25 Auk26 Auk27 Auk28 Auk29 Auk30 Auk31 Auk32 Auk33 Auk34 Auk35 Auk36 Auk37 Auk38 Auk39 Auk40 Auk41

d-ala-d-ala ligase integral membrane sensor kinase d-carboxypeptidase hypothetical, similar to Teg18 Dyp-type peroxidase two-component response regulator d-lactate dehydrogenase NRPS modules 1–2 NRPS module 3 NRPS modules 4–6 NRPS module 7 thymidyltransferase epimerase EvaD methyltransferase EvaC GtfB GtfC aminotransferase EvaB NDP hexose dehydratase EvaA Gtf ABC transporter MbtH-like OxyA OxyE OxyB sulfotransferase halogenase OxyC halogenase b-hydroxylase DAHP synthase transcriptional regulator (StrR like) hydrolase hypothetical (siderophore interacting protein) Hmo transcriptional regulator (LuxR like) membrane ion antiporter HpgT chorismate mutase HmaS Pdh DpgA DpgB DpgC DpgD hydrolase transcriptional regulator (MarR family)

Start [bp]

Stop [bp]

corresponding to the known structure of UK-68,597,[16] except that the structurally similar Hpg amino acid was predicted at position 1 rather than m-chloro-p-hydroxyphenylpyruvate. The NRPS domains for condensation (C), adenylation (A), thiolation (T), epimerization (E), and thioesterase (TE) for peptide chain termination were also predicted. Within the four NRPS open reading frames, the following module arrangement was identified: auk3 encodes modules 1 and 2 (A-T-C-A-T-E), auk4 encodes module 3 (C-A-T-X), auk5 encodes modules 4–6 (C-A-T-E-CA-T-E-C-A-T), and auk6 encodes module 7 (C-A-T-X-TE). This is similar to the module arrangement in the A47934 biosynthetic cluster,[19] where X is a non-functional E domain.

Strand Gene length [bp]

137 2268

1102 1135

+

2341 3124 4917 6092

2934 4857 6011 6784

+ + + +

7852 8313 14564 19111 31360 36974 38724 39953 40226 41515 42860 43978

6800 14567 18673 31326 36972 38044 38089 38739 41449 42732 43981 45372

45466 46730 48833 49155 50353 51497 52735 53672 55241 56541 57885 59794 61487

966 1134 594 1734 1095 693

+ + + +

1053 6255 4110 12216 5613 1071 636 1215 1224 1218 1122 1395

46620 48715 49042 50330 51507 52693 53574 55147 56473 57875 59477 60849 62476

+ + + + + + + + + + + + +

1155 1986 207 1173 1155 1197 840 1476 1233 1335 1593 1056 990

64135 62552 64317 65120

+

1584 804

66286 65141 66978 69401

+

1146 2424

69657 71131 72450 72852 73916 75482 76603 77265 78563 79424 80305

+ + + + + + + + + + +

1362 1320 363 1062 1407 1125 666 1302 801 864 483

71018 72450 72812 73913 75325 76606 77268 78566 79363 80287 80787

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+ + + + +

Nonproteinogenic amino acids Like other GPAs, UK-68,597 has three non-proteinogenic amino acids: Dpg, Hpg, and Bht. All of the known genes encoding the required enzymes for Dpg (DpgA-D)[20] and Hpg (Pdh, HmaS. Hmo, HpgT)[21] biosynthesis could be assigned in the UK-68,597 biosynthetic cluster (Figure 2). The enzymes required for Bht biosynthesis differ between vancomycin- and teicoplanin-type producers.[22] In vancomycin-type producers, Bht is synthesized from Tyr before activation and loading onto the corresponding NRPS A domain.[23] In teicoplanin-type producers, Tyr that is already tethered to the NRPS is hydroxylated by a b-hydroxylase.[24] Homologues in the teicoplanin-type producers are tcp25 (teicoplanin), dbv28 (A40926), and staM (A47934). Two putative b-hydroxylases are present in the UK68,597 cluster: Auk24, with 82 % identity to Tcp25, and another less similar (59 % identity to Tcp25) protein, Auk27, which has a similar conserved domain (DUF2070 superfamily). The limited number of GPA sulfotransferases (Stfs) and glycotransferases (Gtfs) characterized to date modify specific residues on the heptapeptide backbone, and similar enzymes appear to maintain regiospecificity, regardless of the GPA scaffold.[25] The Stf Auk20 and two Gtfs, Auk10 and Auk14, from the UK-68,597 biosynthetic cluster were overexpressed, purified, and tested for their activity with various substrates in vitro (Table 2): vancomycin (10), vancomycin aglycone (8), teicoplanin (6), A47934 (1), and DS-A47934 (3). The associated genes were also conjugated into Streptomyces toyocaensis (A47934 producer) and a DstaL derivative where the sulfotransferase gene is deleted (produces 3). The reaction progress in cell extracts was monitored by reversed-phase high performance liquid chromatography (RP-HPLC) and liquid chromatography mass spectrometry (ESI-LC/MS). The GPAs studied differ in their backbone amino acid sequence and vary in their glycosylation, chlorination, sulfation, and oxidative crosslinking patterns (Table 2). Heptapeptide scaffolds of teicoplanin- and vancomycin-type GPAs differ at amino acids 1 and 3 in that the aromatic residues at amino acids 1 and 3 are replaced by aliphatic residues in vancomycin. Teicoplanin-type GPAs often have a Tyr at amino acid 2, whereas vancomycin-type GPAs often have Bht in this position. Vancomycin and vancomycin aglycone differ in that vancomycin aglycone lacks the disaccharide on amino acid 4. Compound 1 has a teicoplanin-type scaffold but differs from 6 in that ChemBioChem 2014, 15, 2613 – 2623

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Table 2. Relevant GPA chemical structures.

R1

R2

R3

R4

R5

R6

Type

1

SO3

H

H

Cl

H

H

T

2

SO3

H

Cl

H

H

T

3

H

H

Cl

H

H

T

4

H

H

Cl

H

H

T

5

H

SO3

Cl

H

H

T

6 7

H H

H SO3

8

n.a.

n.a.

9

n.a.

10

n.a.

H

H

H H

H

T T

n.a.

n.a.

n.a.

V

n.a.

n.a.

n.a.

n.a.

V

n.a.

n.a.

n.a.

n.a.

V

Compound numbers are shown in the first column. The a-carbons are labeled in the diagrams of teicoplanin (T)- and vancomycin (V)- type scaffolds. n.a.: Not applicable.

1 lacks glycosylation, is sulfated at amino acid 1, and is chlorinated at amino acid 5. Tailoring of the various vancomycinand teicoplanin-type scaffolds occurred with varying efficiencies and led to the synthesis of several novel GPA derivatives as described below. Sulfation One stf gene, auk20, was predicted in the UK-68,597 biosynthetic cluster located between a putative mono-oxygenase and a halogenase (Figure 2). Auk20 is most similar to sulfotransferases TEG13 and TEG14 identified in metagenomic screens,[13] with 48 and 47 % identity, respectively. TEG13 and TEG14 modify the teicoplanin aglycone (but not compound 1) at Dpg3 and Bht6, respectively.[13] To determine the regiospecificity of Auk20 and to explore its potential for turnover of unnatural (non-native) substrates, Auk20 was tested in vitro with vancomycin- (8 and 10) and teicoplanin- (1, 3, and 6) type scaffolds. Although 8, 10, and 1 are not substrates for this enzyme, 3 and 6 are substrates (Table 3). After a 20 h of reac 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Table 3. Turnover of in vitro GPA glycosylation and sulfation reactions.[a]

Auk20 teicoplanin A47934 DS-A47934 vancomycin aglycone vancomycin

[b]

95  3 – 51 18[b] – –

Turnover [%] Auk10 n.d. 24  8 8  4[b] 5.0  0.3 –

Auk14 n.d. – – trace –

[a] n.d.: not determined. –: no product observed. Standard deviation is from three replicates. [b] New compound.

tion, 95 % of 6 was converted to 7, whereas 51 % of 3 was converted to 5 (Figure 3 A, Table 3). The position of Auk20-catalyzed sulfotransfer was determined by isolating 7 from in vitro reactions. The structure was analyzed by HRMS, NMR spectroscopy (Table 4, Figure S5–12), and tandem mass spectrometry (MS/MS). The expected [M H] mass of 7 was 1956.5056, whereas the observed mass ChemBioChem 2014, 15, 2613 – 2623

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Figure 3. In vitro sulfation and glycosylation reactions. A) RP-HPLC (Abs220 nm) of in vitro teicoplanin reactions in the absence (lower trace) or presence (upper trace) of sulfotransferase Auk20. Compounds 6 and 7 are indicated. B) RP-HPLC (y-axis Abs220 nm) of 1 in vitro glycosylation reactions in the absence (lower trace) or presence (upper trace) of glycosyltransferase Auk10. Compounds 1 and 2 are indicated.

was 1956.5015 (D2.1 ppm, Figure S1). Differences in chemical shifts of 0.6, 0.3, and 0.2 ppm (Table 4) were observed for the protons ortho (b, d) and meta (f) to the sulfate group, as reported for teicoplanin aglycone and its sulfated derivative.[12] Compounds 6 and 7 were fragmented by negative ion ESI-MS/ MS using both a QTRAP LC/MS/MS system (Figure S3) and an XL-Orbitrap system (Figure S4). Briefly, fragments shown in Figure S3 represent a loss of 44 amu from the most abundant doubly charged isotopic peak (m/z: [979.9]2 and [937.8]2 for 7 and 6, respectively), giving rise to doubly charged decarboxylated fragments (m/z [958.1]2 and [915.7]2 ), as observed previously with other phenolic compounds.[26] Furthermore, loss of the sugar residue positioned on Bht6 corresponded to m/z values of 1695 and 1611 from 7 and 6, respectively. Upon cleavage of the sugar residue on Dpg7, m/z values of 1534 and 1449 were observed from 7 and 6, respectively. Loss of the acyl chain, followed by the attached sugar, led to frag-

Table 4. Numbering scheme and selected 1H and 13C assignments for compounds 6 and 7 cores in D2O.

Assignment

d 1H (ppm)

6 Multiplicity, J [Hz]

d 13C (ppm)

d 1H (ppm)

Multiplicity, J [Hz]

7 d 13C (ppm)

HMBC

1b 1e 1f 2b 2e 2f 3b 3d 3f 4b 4f 5b 5e 5f 6b 6e 6f 7d 7f

6.82 6.95 7.10 7.23 6.99 7.36 6.34 6.08 6.52 5.55 5.07 6.81 6.80 6.84 7.61 7.29* 7.18* 6.56 6.69

m, 1 H d, 1 H, 8.1 d, 1 H, 8.1 s, 1 H d, 1 H, 8.1 d, 1 H, 8.1 s, 1 H s, 1 H s, 1 H s, 1 H s, 1 H m, 1 H m, 1 H m, 1 H s, 1 H d, 1 H, 8.1 d, 1 H, 8.1 s, 1 H s, 1 H

119.8 117.8 126.5 131.2 123.8 129.1 108.4 103.9 108.7 107.4 103.8 134.8 117.0 126.0 128.4 123.5 127.3 105.5 102.4

6.65 6.73 6.95 7.24 7.05 7.35 6.94 6.40* 6.67* 5.44 5.19 6.80 6.68 6.80 7.62 7.31 7.17 6.36 6.55

s, 1 H d, 1 H, 8.1 m, 1 H s, 1 H d, 1 H, 8.1 d, 1 H, 8.1 m, 1 H s, 1 H s, 1 H br s, 1 H br s, 1 H m, 1 H m, 1 H m, 1 H s, 1 H d, 1 H, 8.1 d, 1 H, 8.1 s, 1 H br s, 1 H

119.6 117.3 125.1 131.3 123.8 129.2 111.4 109.5* 113.8* 106.6 104.3 134.8 120.7 125.9 128.6 123.7 127.1 110.4 103.2

125.1 125.1 119.6 129.2; 149.5 129.2; 149.5 131.3; 149.5 – 111.4; 113.8 109.5; 111.4 104.3; 132.9 132.9 117.6;120.7; 125.9 125.9 117.6;120.7 127.1; 149.3 127.1; 149.3 123.7; 128.6; 149.3 103.2; 117.5 110.4; 117.5

R2 is H (6) or a sulfate group (7). *: assignments may be reversed. The chemical shifts of Dpg3 are shown in bold.

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CHEMBIOCHEM FULL PAPERS ments with m/z values of 1296 and 1134 from 6 and an m/z value of 1379 from 7. Similar fragmentation patterns were observed when compounds were fragmented using the XL-Orbitrap system (Figure S4). MS/MS results are summarized in Figure S2 and Table S1. Further analysis of the sulfated compound by 1H NMR showed clear deshielding of the aromatic protons of Dpg3. Taken together, ESI-MS/MS and NMR data are consistent with sulfation of compound 6 by Auk20, occurring at Dpg3-producing compound 7, where it has been reported with UK-68,597, implicating Auk20 as the sulfotransferase involved in UK-68,597 biosynthesis. In addition to in vitro studies, we created an in vivo platform to genetically engineer S. toyocaensis with GPA tailoring enzymes from newly sequenced clusters. Each tailoring enzyme was cloned into the pSET152 vector, engineered with the constitutive promoter (ermEp*)[27] for integration into both the wild-type and the DstaL deletion mutant of S. toyocaensis, which produce compound 1 and 3, respectively. Chromosome integration was confirmed by PCR (data not shown), and strains were examined for analogue production. Results of fermentation were consistent with in vitro results, showing sulfation of compound 3 but not 1. Fermentations of S. toyocaensis DstaL ermEp*::auk20 resulted in the production of compound 5 (Figure S13), which is the expected product of compound 3 sulfated at amino acid 3. A disulfated form of 1 was not observed from S. toyocaensis ermEp*::auk20 fermentations. Glycosylation Interestingly, UK-68,597 is glycosylated at position 4 with glucose and l-vancosamine,[28] but three predicted glycosyltransferases are found in its biosynthetic cluster: Auk10, 11, and 14. Other GPAs studied to date with disaccharides, such as vancomycin and chloroeremomycin, require one Gtf per monosaccharide unit added to the GPA scaffold.[22] Vancomycin has the same disaccharide at Hpg4 as UK-68,597: l-vancosamine 1,2glucose. The vancomycin biosynthetic cluster has two glycosyltransferases, GtfE and GtfD, which respectively catalyze attachment of the glucose and vancosamine sugars.[10] When compared to other GPA Gtfs, Auk10 and Auk11 enzymes grouped according to predicted regiospecificity. Auk10 is most similar to Gtfs that add the first sugar moiety to Hpg4 of the heptapeptide scaffold, such as tGtfB of the teicoplanin biosynthetic cluster (73 % identity)[29] and GtfE of the vancomycin biosynthetic cluster (73 % identity).[11] This is comparable to the 82 % and 68 % identity between GtfBcep and tGtfB to GtfE, respectively. Auk11 showed similarity to enzymes that transfer an additional sugar moiety to a heptapeptide scaffold glucosylated at Hpg4, resulting in a disaccharide at Hpg4. Auk11 has 71 and 70 % identity to GtfC and bGtfC, which add l-4-epi-vancosamine and dehydrovancosamine to chloroeremomycin and balhimycin, respectively.[11, 22] The similarity of these enzymes to GtfB and GtfE led us to predict that Auk10 glucosylates Hpg4 of the UK-68,597 heptapeptide scaffold and that Auk11 transfers l-vancosamine to synthesize l-vancosamine-1,2-glucose. Auk14 is most similar to Gtfs that add various sugar moieties to amino acid 6 with 64 % identity to tGtfA (NCB accession  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chembiochem.org number CAE53349.1), which adds N-acetyl-glucosamine to teicoplanin at Bht6 and 63 % identity to the GtfAcep (GenBank accession number AAB49292.1), which adds 4-epi-vancosamine to Bht6 of chloroeremomycin. However, Auk14 is also 59 % identical to the GtfBcep and 62 % identical to Auk10. The similarity of the extraneous Gtf, Auk14, to GtfA enzymes is quite interesting, as UK-68,597 has no sugar on amino acid 6. As both Auk10 and Auk14 showed some similarity to enzymes that transferred glucose to Hpg4 of the heptapeptide, the glucosylating activity of both enzymes was examined in vitro. UK-68,597 is glycosylated at Hpg4, an amino acid shared between vancomycin- and teicoplanin-type GPAs. Enzymes were tested with both types of scaffolds to examine their regiospecificity and substrate promiscuity and to investigate their potential for generating novel GPAs. Gtfs were tested in vitro using the following substrates: A47934 (1), DS-A47934 (3), vancomycin aglycone (8), and vancomycin (10). Auk10 was able to use UDP-glucose as a substrate when reacted with 1, 3, and 8 with 24, 8, and 5 % turnover, respectively, after twohour incubations (Figure 3, Table 3). Similarly, Auk14 was able to use UDP-glucose as a substrate, with 8 producing trace amounts of glycosylated product that were detectable by LCMS. Compound 10 was not a substrate for either Auk10 or Auk14. As Auk10 had a much higher glucosylating activity than Auk14, the regiospecificity of Auk10 was further investigated. Regiospecificity was determined by using a combination of techniques. In vitro, Auk10 glycosylated 1 in the presence of UDP-glucose (Figure 3), resulting in 2. The structure of 2 was analyzed by HRMS, NMR, and LC/MS-MS. The expected [M H] mass of 2 was 1472.1832, however, the observed mass was 1472.1855 (D1.6 ppm, Figure S1). The NMR spectrum (Table 5, S14–18) of 2 was consistent with its predicted structure, showing key HMBC correlations between Hpg4 and glucose (Table 5). Compounds 1 and 2 were fragmented by negative ion ESI-MS/MS using both a QTRAP LC/MS/MS system (Figure S20) and an XL-Orbitrap system (Figure S21). Upon fragmentation by negative ion ESI-MS/MS, 1 and 2 produced two sets of distinct daughter ions (Figure S20). In fragmentation analyses of 1 and 2, identical losses of 44 amu from doubly charged parent ions gave rise to m/z values of [633.9]2 and [714.4]2 , respectively. We observed losses of 404 amu from both 1 and 2, corresponding to losses of Hpg5 and Dpg7, as reported previously,[12] that give rise to m/z values of 906.9 from 1 and 1069.9 from 2, its glycosylated analogue, clearly indicating that there is no glucose on residues 5 or 7. Similar fragmentation patterns were observed when compounds were fragmented using the XL-Orbitrap system (Figure S21). MS/MS spectra are summarized in Figure S20 and Table S2). Together with the observation that the enzyme glucosylates 8 but not 10 in vitro, this suggests that Auk10 glycosylates at position 4, as this is the only available phenolic hydroxyl group. The gtf genes auk10 and auk14 were also conjugated into S. toyocaensis and its DstaL derivative, as described for auk20. Glycosylated products were not observed in the fermentations of either the wild-type or DstaL strains.

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Table 5. Numbering scheme and 1H and 13C assignments for compound 2 in [D6]DMSO.

Assignment

1[44] d 1H (ppm)

d 1H (ppm)

Multiplicity, J [Hz]

2 d 13C (ppm)

COSY

HMBC

1b 1e 1f 2b 2e 2f 3b 3d 3f 4b 4f 5b 5f 6b 6e 6f 7d 7f 1’ 2’ 3’ 4’ 5’ 6’1 6’2

6.57 7.65 7.12 7.20 7.16 7.68 6.35 6.30 6.38 5.67 5.03 7.24 6.78 7.63 7.19 7.45 6.26 6.37 n.a. n.a. n.a. n.a. n.a. n.a. n.a.

6.58 7.65 7.12 7.21 7.20 7.68 6.35 6.30 6.36 5.68 5.09 7.25 6.78 7.64 7.25 7.45 6.28 6.37 5.33 3.35 3.29 3.20 3.25 3.69 3.49

br s, 1 H m, 1 H m, 1 H s, 1 H m, 1 H d, 1 H, 8.1 s, 1 H s, 1 H s 1H s, 1 H s, 1 H s, 1 H s, 1 H s, 1 H m, 1 H d, 1 H, 8.1 brs, 1 H s, 1 H d, 1 H, 7.6 m, 1 H m, 1 H m, 1 H m, 1 H d, 1 H, 10.1 dd, 1 H, 10.8, 4.4

116.5 127.2 124.1 130.8 125.1 130.6 105.9 111.3 105.9 109.6 105.4 134.9 125.4 127.2 123.4 127.7 106.4 102.5 102.7 74.1 76.7 70.1 77.5 61.2 61.2

7.12w 7.12 7.65 – 7.68 7.20 – – – – – 6.78w 7.25w 7.45w 7.45 7.25, 7.64w – – 3.35 5.33, 3.29 3.20, 3.35 3.25, 3.29 3.49, 3.20 3.49, 3.25w 3.69, 3.25

143.7, 124.1w 143.7 – 151.3, 130.6 151.3, 135 151.3, 130.8 111.3, 157.5 105.9, 157.5 111.3, 157.5 55.1, 105.4, 133.2, 152.0 55.1, 109.6, 133.2, 152.0 53.1, 125.4, 151.6 53.1, 134.9, 151.6 72.1, 127.7, 148.2 127.2, 148.2 72.1, 127.2, 148.2 102.5, 116.3 106.4, 116.3 77.5, 133.2 102.5, 76.7 70.1, 74.1 61.2, 77.5 74.1 70.1 77.5

Compound 1 in [D6]DMSO 1H assignments are as reported previously.[44] n.a.: not applicable.

Self-resistance and other features of UK-68,597. GPA resistance in antibiotic producers is mediated by the vanHAX resistance gene cassette, which modifies the drug target, the bacterial cell wall, to terminate with d-Ala-d-Lac instead of the canonical d-Ala-d-Ala. The vanHAX genes were identified on a separate contig surrounded by peptidoglycan biosynthetic genes (GenBank accession number KF219715). Separation of the vanHAX cassette from the GPA biosynthetic cluster has also been observed in the balhimycin producer A. balhimycina.[30] Interestingly, the UK-68,597 cluster possesses other known resistance-associated genes within the cluster, including a d-Ala-d-Ala ligase (Ddlauk) and a potential resistance cassette organized as follows: vanSYxxRH, where x represents a gene of unknown function (Figure 2).  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Lastly, the UK-68,597 cluster has two putative halogenases, and the molecule has four sites of chlorination (Figure 2). Auk21 shows 92 and 85 % identity to the putative halogenases of the teicoplanin and chloroeremomycin biosynthetic clusters, respectively. UK-68,597, teicoplanin, and chloroeremomycin are all chlorinated at Tyr/Bht2 and Bht6. Auk23 is more distantly related, showing 70 and 66 % identity, respectively, to the same halogenases. Further analysis is required to determine the regiospecificity of the UK-68,597 halogenases Auk21 and Auk23.

Discussion Genome sequencing is becoming a rapid and cost-effective strategy for identifying biosynthetic clusters.[31] In the case of ChemBioChem 2014, 15, 2613 – 2623

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CHEMBIOCHEM FULL PAPERS this clinically important class of antibiotic, biosynthetic clusters can be further mined for their tailoring enzymes to expand chemical diversity by reacting them with different natural and “unnatural” substrates. Derivatization of traditional antibiotic scaffolds has been a successful strategy for increasing the clinical usefulness of many classes of antibiotics, such as b-lactams and macrolides. It is essential that we increase the pool of potential derivative glycopeptides and other antibiotics from which those with clinically favorable characteristics can be selected. This study contributes the tailoring enzymes of the 81 kb UK-68,597 biosynthetic cluster to the “toolbox” of enzymes which can be used to modify existing GPA scaffolds. GPA sulfation is found less frequently than other modifications, such as glycosylation or chlorination. However, metagenomic studies with environmental samples have recently significantly expanded the number of known GPA sulfotransferases, which now number six gene products: TEG12, TEG13, TEG 14, AZ205 sulfotransferase, StaL, and Pek25.[12, 13, 15, 32] Three Stfs (TEG13, 14, and AZ205) associated with teicoplanin-type sulfation transfer a sulfate to residues common to both vancomycin- and teicoplanin-type GPAs and can potentially be used to derivatize both types of scaffolds. Sulfation by TEG13 on amino acid 6 (Bht) and TEG14 on residue 4 (Hpg) occurs where glycosylation is often documented in other GPA antibiotics. It is interesting that Auk20 was able to modify a non-sulfated GPA (compound 3) but was not able to induce turnover of the related sulfated GPA (compound 1). The active site of Auk20 might not be able to accommodate compound 1, as it has a negatively charged sulfate at amino acid 1. Resolution of this question will require determination of the atomic structure of Auk20 in the presence and absence of substrates. Structures of other sulfotransferases have been informative in establishing substrate specificity but only one with the natural substrate (compound 3 in StaL) bound in the active site.[33] This shows conformational flexibility of the scaffold, but more importantly, the molecule extends out of the binding site of one subunit and interacts in the adjacent binding site of the second subunit. Ultimately, it is difficult to predict which Stfs will show more substrate promiscuity when sulfating a GPA that is already tailored at a position that is “unnatural” for all of the respective enzymes without additional detailed structural studies. Sulfation might be an important GPA modification useful in the synthesis of novel GPAs with the ability to evade resistance. In the case of 1, it is known that addition of sulfate (at amino acid 1) causes a modest (twofold) increase in the MIC value[32] but evades induction of vancomycin resistance by dampening transcription activation of resistance genes vanHAX via the VanRS two-component system. This highlights the importance of expanding the number of Stfs from which tailoring enzymes can be chosen. GPA Stfs need to be further explored for both their regiospecificity and their substrate promiscuity, so that candidates that are able to efficiently utilize different GPA scaffolds (both teicoplanin and vancomycin scaffolds with different tailoring) at the appropriate amino acid residues can be identified. The most promising Stfs could be the starting points for enzyme evolution, with the goal of increasing sub 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chembiochem.org strate promiscuity and catalytic efficiency. The addition of Auk20, the UK-68,597 sulfotransferase that modifies residue 3, to this toolkit provides an additional catalyst for the expansion of GPA chemical diversity. Auk10 is a Gtf shown to efficiently transfer a glucose molecule to amino acid 4 of the heptapeptide scaffold. Similarity to several characterized enzymes suggests that Auk11 attaches vancosamine to glucose. Auk14 is similar to known GPA Gtfs that add a sugar group to the amino acid at position 6. As UK68,597 has no sugar on amino acid 6, Auk14 might add a sugar moiety to position 6 in a minor product of the biosynthetic cluster (as yet undetected). Precedent for this hypothesis can be found in the balhimycin biosynthetic cluster, in which there are three Gtfs but only two glycosylations in the major product, balhimycin. The third Gtf is proposed to glycosylate minor products of biosynthesis, balhimycin V and dechlorobalhimycin V, which have three glycosylations.[34] Alternatively, activity might not have been observed with Auk14 because a TDP sugar, rather than UDP-glucose, is the preferred substrate. A thymidyltransferase in the chloroeremomycin GPA biosynthetic cluster has been characterized,[35] and another is predicted (Auk7) in the UK-68,597 cluster. The chloroeremomycin thymidyltransferase has been shown to catalyze the formation of TDP-glucose from glucose-1-phosphate and dTTP.[35] However, previous studies with other GPA Gtfs show no preference for TDP-sugars over their UDP-sugar equivalents.[10] Gtfs studied to date have shown a wide range of substrate promiscuity in terms of the sugar moiety[7–9] and the GPA scaffold utilized by the enzyme. For example, when the three Gtfs of vancomycin were systematically examined with a variety of NDP-sugars, trace (kcat < 0.001 min 1) to good (kcat = 135 min 1) transfer of sugar moieties to vancomycin aglycone was observed.[9] Similarly, when different Gtfs were examined for their tolerance for natural and “unnatural” GPA substrates, results also varied widely, with kcat values from  0.3 min 1 to 60 min 1.[11] For example, GtfBcep efficiently glucosylates natural substrate vancomycin aglycone but had very low glucosylation activity for an alternate substrate, teicoplanin aglycone. Comparatively, the vancomycin GtfE was much more efficient at glucosylating both natural and “unnatural” substrates: vancomycin aglycone and teicoplanin aglycone.[11] The timing of the events involved in GPA biosynthesis has been recently reviewed.[22] Briefly, biosynthesis begins with formation of the nascent peptide chain at the NRPS. With the exception of Bht biosynthesis in teicoplanin-type producers, the non-proteinogenic amino acids and usual sugars are synthesized before or “just in time” for addition to the GPA scaffold. Oxidative crosslinking then occurs as the peptide is elongated. Glycosylation then occurs on the fully crosslinked GPA. It is unclear if sulfation occurs before or after glycosylation. In this study, the order of sulfation and glycosylation was not further clarified, as sulfation and glycosylation were shown on glycosylated/non-glycosylated and sulfated/non-sulfated substrates, respectively. Consideration must also be given to the fact that “unnatural” substrates were also used in this study. With the exception of complestatin (a non-antibiotic natural product with some structural similarity to GPAs), characterized ChemBioChem 2014, 15, 2613 – 2623

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glycopeptides with reported biosynthetic clusters (vancomycin, chloroeremomycin, balhimycin, A47934, teicoplanin, and A40926)[22] have two or three chlorination sites. Molecules with two chlorination sites usually possess one halogenase in the biosynthetic cluster, whereas halogenation on three amino acids (A47934, complestatin) is likely encoded by two halogenases.[19, 36] Chlorination has been reported as an important modification for activity,[37] but the effects of different sites and multiple chlorination events have not been elucidated. The presence of an a-keto acid at the N-terminus is unusual for a GPA and unique to UK-68,597. During biosynthesis, this might occur by the activation of p-hydroxybenzoylformate by the A domain of the NRPS module 1 instead of Hpg. Alternatively, as with other GPAs, Hpg might be recognized by module 1 and incorporated into the elongating peptide, and additional enzymes might act to convert the amino acid to the a-keto acid. Interestingly, p-hydroxybenzoylformate is an intermediate of Hpg biosynthesis.[21] HpgT is a transaminase that transforms p-hydroxybenzoylformate to Hpg. If p-hydroxybenzoylformate is incorporated into the growing peptide chain, the predicted peptide backbone will be consistent with the backbone of UK-68,597 without the need of any additional modifications. The second hypothesis is supported by the ability of A domain specificity predictor programs, such as those present in antiSMASH,[18] to predict amino acid 1 as Hpg rather than an unknown amino acid.

from an organism with poor antibiotic production. The addition of a new collection of GPA tailoring enzymes from the UK68,597 biosynthetic cluster enriches our cadre of GPA-modifying catalysts essential to expanding chemical and functional diversity in this class.

Conclusions

Isolation of genomic DNA and 454 pyrosequencing: Actinoplanes sp. ATCC 53533 genomic DNA was isolated by using a salting out procedure. Briefly, cells from culture (50 mL) were resuspended in lysis buffer (5 mL, Qiagen DNeasy kit; 20 mm Tris pH 8.0, 2 mm EDTA, 1.2 % Triton X-100, v/v) and SET buffer (5 mL).[39] The cells were homogenized with a glass homogenizer, collected by centrifugation, and resuspended in lysis buffer (10 mL). Lysozyme (25 mg mL 1) and mutanolysin (10 mg mL 1) were added to the cell suspension, followed by incubation at 37 8C overnight. After the overnight incubation, proteinase K (4 mg) and 10 % sodium dodecylsulfate (600 mL) were added to the emulsion and incubated at 55 8C for 3 h. NaCl (5 m, 5 mL) was added and mixed thoroughly by inverting the tube. Chloroform (10 mL) was added, and the emulsion was incubated at room temperature on a rocking platform for 30 min. The solution was centrifuged at 4000 g for 15 min, and the upper aqueous phase was transferred to a 50 mL Falcon tube. Isopropanol (6 %, v/v) was added and mixed by inversion to precipitate the DNA. Genomic DNA was spooled using a sealed Pasteur pipette and washed in 70 % ethanol. After air drying, the DNA was dissolved in water (200–500 mL). The genomic DNA yield and quality were assessed using a NanoDrop spectrophotometer and submitted for library preparation and sequencing on a Roche 454 pyrosequencer.

Glycopeptide antibiotics are important in the treatment of Gram-positive infections. However, antibiotic therapy is becoming less effective, due to the emergence of drug resistance. Glycopeptides are built from core heptapeptide backbones that are enzymatically tailored with oxidative cross-linking, glycosylation, sulfation, halogenation, acylation, methylation, etc. at different positions at varying frequencies, generating extensive chemical and functional diversity. However, the physiological impact of each modification has not been well studied, although at least in the case of sulfation, modification is associated with attenuated resistance. Systematic characterization of each tailoring enzyme, including regiospecificity and substrate flexibility, can increase chemical diversity, which lends itself to hypothesis- and activity-driven drug design. Tailoring enzymes constitute a “toolbox” to creatively accessorize the GPA scaffold in a manner remnant of nature to build novel natural products. Actinoplanes sp. ATCC 53533 is a complex bacterium that is not readily amenable to genetic manipulation and is a poor producer of UK-68,597. The producer was sequenced, and the glycopeptide biosynthetic cluster was identified in a draft genome assembly. Two orthogonal approaches were then elected to study the biosynthetic cluster: isolation and in vitro characterization of selected enzymes, as well as integration of the genes into a new host, S. toyocaensis, the producer of a GPA aglycone, A47934. This study expands the pool of GPAs available and provides a workflow to produce more derivatives from which those with desirable features can be selected, even  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Experimental Section Bacterial strains, culture methods and reagents: Lysogeny broth (LB; Lennox) or agar plates were used for growth and maintenance of cloning strains Escherichia coli TOP10 (Invitrogen) at 37 8C, E. coli Rosetta(DE3) (Novagen) at 37 8C, and Rhodococcus erythropolis L88[38] at 30 8C. S. toyocaensis and Actinoplanes sp. were routinely grown on Streptomyces antibiotic medium (SAM),[19] SAM supplemented with 1.5 % agar, or Bennett’s agar[39] at 30 8C. All antibiotics and media components were obtained from Sigma–Aldrich or Bioshop Canada (Burlington, ON). Media were supplemented with chloramphenicol (35 mg mL 1), kanamycin (25 mg mL 1), and apramycin (25 mg mL 1) as appropriate. Actinoplanes was fermented in several media : V6 (2 % glucose, 0.5 % yeast extract, 0.5 % beef extract, 0.3 % casein hydrosylate, 0.5 % bacto peptone, 0.15 % NaCl, pH 7.3), AF/MS (2 % glucose, 0.8 % soybean meal, 0.2 % yeast extract, 0.1 % NaCl, 0.4 % CaCO3, pH 7.3), M8 (1 % glucose, 2 % soluble starch, 0.4 % casein hydrosylate, 0.2 % yeast extract, 0.2 % beef extract, 0.3 % CaCO3, pH 7.2), and media described in the original patent[40] (1 % cerelose, 1 % corn starch, 1 % soya flour, 0.5 % distillers solubles, 0.5 % NaCl, 0.1 % CaCO3, 0.0002 % CoCl2, pH 6.9–7.0).

Library preparation was performed by the McMaster Metagenomic Center as described previously.[38] Reads were assembled into contigs with MIRA using the “genome, accurate, denovo, 454” settings (version 3.4.1,[41]). The resulting 13.2 Mb of consensus sequence in 714 contigs (N50 59320 bp) was searched with translated BLAST[42] using the teicoplanin biosynthetic gene cluster as a query (GenBank accession number AJ632270) to identify a sequence containing a putative 80.8 kbp GPA biosynthetic cluster (Table 1) and a separate vanHAX resistance cluster (GenBank accession number KF219715). ChemBioChem 2014, 15, 2613 – 2623

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Overexpression and isolation of recombinant proteins: Genes encoding the three tailoring enzymes auk10, auk14, and auk20 were PCR-amplified from Actinoplanes sp. ATCC 53533 genomic DNA by using primers engineered with restriction endonuclease sites: Auk10 F NdeI/Auk10 R EcoRI, Auk14 F2 NdeI/Auk14 R3 XhoI, Auk20 F NdeI/Auk20 R EcoRI, respectively. Oligonucleotide sequences are listed in Table S1.

(0.5 mm; DS-A47934, A47934, vancomycin, or vancomycin aglycone), and enzyme (10 mm) were incubated for 2 h at 37 8C. Largescale (10 mL) reactions contained enzyme (5 mm) and were incubated for 2 h at 37 8C, supplemented with additional enzyme (5 mm) at 2 h to a final enzyme concentration of 10 mm and incubated for an additional 2 h.

PCR fragments containing auk20 and auk10 were digested with NdeI and EcoRI and cloned into pET28a (Novagen) and pTIPQC1,[43] respectively. PCR products containing auk14 were digested with NdeI and XhoI and cloned into pTIPQC2.[43] Plasmids were routinely maintained in E. coli TOP10 (Invitrogen).

Reaction progress was monitored by RP-HPLC or ESI-LC/MS. Reactions were stopped with an equal volume of cold 100 % CH3CN. Precipitate was removed by high-speed centrifugation for 10 min.

Plasmids containing auk10 and auk14 were transformed into R. erythropolis L-88 by electroporation, expression was induced, the cells were lysed, and the supernatant was clarified, loaded onto NiNTA (Qiagen) columns, and eluted as described previously[38] with the following exceptions: cultures for protein expression were grown at 16 8C for 3 days post-induction; the composition of buffer A was HEPES (50 mm, pH 8.5), NaCl (500 mm), imidazole (1 mm), 5 % glycerol (v/v), and the composition of buffer B was HEPES (50 mm, pH 8.5), NaCl (500 mm), imidazole (250 mm), and 5 % glycerol (v/v). Plasmids containing auk20 were transformed into chemically competent E. coli Rosetta(DE3) (Novagen) prepared by standard protocols. A single colony transformant was used to inoculate a 10 mL LB starter culture supplemented with chloramphenicol and kanamycin. The culture was grown overnight and diluted 1:100 into LB (1 L) supplemented with chloramphenicol and kanamycin. The diluted culture was grown to an OD600 of 0.4, at which time IPTG was added to a final concentration of 1 mm. Cultures were grown for an additional 16 h at 16 8C before harvesting. The cells were lysed, and the supernatant was clarified, loaded onto Ni-NTA (Qiagen) columns, and eluted as described above, except that lysozyme was used at a final concentration of 0.5 mg mL 1. Proteins were concentrated and desalted using Amicon centrifugal filter units (Millipore), and the following buffer: HEPES (50 mm, pH 8.5), NaCl (200 mm), dithiothreitol (DTT, 2 mm), and 5 % glycerol (v/v). After concentration, proteins were analyzed by SDS-PAGE, quantified using the Bio-Rad protein assay, and glycerol was added to a final concentration of 10 % (v/v) and stored at 80 8C for later use. Purification of GPA antibiotics: A47934 and derivatives were purified from S. toyocaensis and S. toyocaensis DstaL as described previously.[14, 32] Vancomycin aglycone was prepared by acid hydrolysis. Briefly, 50 mg vancomycin was dissolved in trifluoroacetic acid (2 mL) and incubated at 40 8C for 3 h. The reaction was diluted 10  in water and dried by lyophilization, followed by purification on a 13 g reversed-phase C18 flash column (Teledyne Isco, Lincoln, NE, USA). In vitro enzymatic assays: Small-scale (50–100 mL) sulfotransferase reactions contained HEPES (50 mm, pH 7.5), DTT (0.1 mm), 3’-phosphoadenosine-5-phosphate (PAPS; 0.45 mm), GPA (0.05 mm; DSA47934, A47934, teicoplanin, vancomycin, or vancomycin aglycone), and enzyme (5 mm). Large-scale (10 mL) reactions contained HEPES (50 mm, pH 7.5), DTT (0.1 mm), PAPS (0.3 mm), GPA (0.4 mm; DS-A47934, A47934, or teicoplanin), and enzyme (5 mm). Reaction mixtures were incubated at 37 8C for 20 h. Small-scale (50–100 mL) in vitro GPA glycosyltransferase reactions containing TAPS (50 mm, pH 8.4), MgCl2 (10 mm), DTT (1 mm), bovine serum albumin (1 mg mL 1), UDP-glucose (5 mm), GPA  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

RP-HPLC and LC/MS analysis: Analytical RP-HPLC was carried out on a Waters 2998 photodiode array detector and an e2695 separation module. An Atlantis T3 5 mm C18 column (4.6  100 mm) was used for separation of glycosylated products, and an XSelect CSH 5 mm C18 column (4.6  100 mm) was used for separation of sulfated products. Separation was performed using a flow rate of 1 mL min 1 under a 10–45 % linear gradient of CH3CN with 0.05 % trifluoroacetic acid (solvent B) in water with 0.05 % trifluoroacetic acid (solvent A) over 7 min. Semi-preparative RP-HPLC separation was carried out using a flow rate of 4 mL min 1 with the gradients listed previously. For isolation of teicoplanin sulfate, a linear gradient of 30–35 % B over 8 min was used on an XSelect CSH 5 mm C18 column (10  100 mm). Teicoplanin sulfate-containing fractions were lyophilized and further separated using the same gradient on an Atlantis T3 5 mm C18 column (10  100 mm). Glycosylated A47934 was isolated using a linear gradient of 10–70 % B over 9 min on an XSelect CSH 5 mm C18 column (10  100 mm). LC-ESI-MS data were obtained using an Agilent 1100 Series LC system (Agilent Technologies Canada, Inc.) and a QTRAP LC/MS/MS System (Applied Biosystems). RP-HPLC was performed using a C18 column (SunFire C18 5 mm, 4.6  50 mm, Waters) with an Agilent 1100 LC binary pump at a flow rate of 1 mL min 1, under the following conditions: isocratic 5 % solvent B (0.05 % formic acid in CH3CN) and 95 % solvent A (0.05 % formic acid in water) for 1 min, followed by a linear gradient to 97 % B over 6 min. NMR, HRMS, and MS/MS: Compound structures were confirmed by 1D and 2D NMR experiments on a Bruker AVIII 700 MHz instrument equipped with a cryoprobe. NMR experiments included 13C distortionless enhancement by polarization transfer, including the detection of quaternary nuclei (DEPTQ), 1H,1H COSY, 1H,13C HSQC, and 1H,13C HMBC. Compound 2 was dissolved in [D6]DMSO; compounds 6 and 7 were dissolved in D2O containing 0.3 % NH4OH. Chemical shifts are reported in parts per million relative to tetramethyl silane using the residual solvent signal as an internal signal. HRMS and MS/MS were obtained using a Thermo Fisher XL-Orbitrap Hybrid mass spectrometer (Thermo-Fisher, Bremen, Germany) equipped with electrospray interface operated in negative ion mode. LC/MS/MS analysis was performed on a QTRAP 2000 (Applied Biosytems) in negative ESI mode. The conditions for LC/MS/ MS analysis were as follows: capillary voltage 4500 eV, declustering potential 30 eV, entrance potential 10 eV, collision energy 10 eV or 25 eV, source temperature 100 8C, interface heater on. The mass range was set from 500 to 1700 Da. The instrument was set to allow passage of [M 2 H]2 ions (sample concentration of 0.1 mg) through the Q1 detector, and ion fragments were scanned in Q3. ChemBioChem 2014, 15, 2613 – 2623

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