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Journal of Bacteriology, April 2009, p. 2871-2875, Vol. 191, No. 8
0021-9193/09/$08.00+0     doi:10.1128/JB.01747-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Modulation of Deoxysugar Transfer by the Elloramycin Glycosyltransferase ElmGT through Site-Directed Mutagenesis

Angelina Ramos, Carlos Olano, Alfredo F. Braña, Carmen Méndez, and José A. Salas^*

Departamento de Biología Funcional e Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Universidad de Oviedo, 33006 Oviedo, Spain

Received 15 December 2008/ Accepted 11 February 2009

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The glycosyltransferase ElmGT from Streptomyces olivaceus is involved in the biosynthesis of the antitumor drug elloramycin, and it has been shown to possess a broad deoxysugar recognition pattern, being able to transfer different L- and D-deoxysugars to 8-demethyl-tetracenomycin C, the elloramycin aglycone. Site-directed mutagenesis in residues L309 and N312, located in the /β/ motif within the nucleoside diphosphate-sugar binding region, can be used to modulate the substrate flexibility of ElmGT, making it more precise for transfer of specific deoxysugars.

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Natural products produced by microorganisms are frequently glycosylated. Bioactivity of many of these compounds is frequently dependent upon the regio- and stereospecific attachment of the sugar moieties (25). Attachment of the sugars to aglycone scaffolds usually occurs in late stages of biosynthesis by the action of glycosyltransferases (GTFs). The acceptor substrates of these GTFs vary widely, but the donor substrates are generally activated deoxynucleoside diphosphate (dNDP) sugars, mainly dTDP-hexoses (8, 23, 24). Over the last few years, increasing evidence has demonstrated a certain degree of flexibility in GTFs regarding the acceptor substrate (7, 12, 26, 27) and, most frequently, the donor substrate (2, 5, 17, 22). X-ray structures of some GTFs involved in antibiotic biosynthesis have been reported: GtfA, GtfB, and GtfD in glycopeptide biosynthesis (14-16); AviGT4 in avilamycin A biosynthesis (11); UrdGT2 in urdamycin A biosynthesis (13); and CalG34 in calicheamicin biosynthesis (28). In addition, the structures of two other GTFs involved in antibiotic biosynthesis and resistance have been reported (3). All of them belong to the GT-B superfamily of GTFs, and they share a two-domain structure, each adopting an /β motif similar to a Rossmann fold. One of these domains would be responsible for aglycone binding (N-terminal domain) and the other for dNDP-sugar binding (C-terminal domain).

The ElmGT glycosyltransferase catalyzes one of the late steps in the biosynthesis of the anthracycline-like antitumor drug elloramycin in Streptomyces olivaceus Tü2353. It transfers an L-rhamnose moiety from dTDP-L-rhamnose to the elloramycin aglycone 8-demethyl-tetracenomycin C (8DMTC) (Fig. 1A) (2). ElmGT is an interesting GTF since it possesses remarkable donor substrate flexibility. It has been shown to transfer at least 11 different sugars to 8DMTC, including both L- and D-isomeric forms of some sugars (4, 9, 18, 19, 21). This surprising flexibility of ElmGT prompted us to get further insight into the identification of some amino acid residues involved in the selection of the sugar donor.

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FIG. 1. (A) Structures of elloramycin and 8DMTC. (B) Sequence alignments of selected glycosyltransferases showing the conserved region including the /β/ motif and the secondary structure of this region. (C) Tertiary structure of GTF GtfB as determined by X-ray analysis (14) and prediction of ElmGT structure using the SWISS-MODEL Workspace (1) showing the Nβ5-N5 loop proposed to be the acceptor binding site and the /β/ motif included in the donor binding site (6). Amino acids L309 and N312, targeted by site-directed mutagenesis in ElmGT in this paper, and their counterparts in GtfB (M330 and Q333) are indicated in boldface.

One of the common features of the antibiotic GTFs so far characterized by X-ray analysis is the existence of a conserved sequence motif located closed to the C terminus of these transferases (Fig. 1B). This motif is present in all eight GTFs characterized so far and can also be found in the primary structure of a number of GTFs that have been cloned and sequenced. This sequence motif has been suggested to be involved in binding the glycosyl donors (6). There are a number of well-conserved residues in this motif, some of which have been shown to interact with different atoms of the nucleotide sugar donor (11, 15, 16). In the absence of information on the structure of ElmGT, we took advantage of the available data from the X-ray structure of the eight antibiotic GTFs reported and the fact that modeling of ElmGT based on these structures keeps the common structural pattern all along the protein. It has been suggested that the poorly conserved loop connecting the Cβ5 strand with the C5 helix could participate in binding and recognition of the sugar, possibly being a characteristic of inverting GTFs (6, 11). This region has been named the /β/ motif (Fig. 1B and C). Comparison of the amino acids in this loop in different GTFs (Fig. 1B) shows this region is quite variable. Only an aspartic acid (D) residue is well conserved, which is sometimes substituted for by a glutamic acid (E). Mutations in this aspartic acid residue have been shown to greatly reduce the catalytic activity of GtfB, thus involving this residue in the catalytic apparatus of this GTF (14). In ElmGT, the /β/ motif contains five residues (amino acids A308, L309, A310, D311, and N312). Based on all of these data, we decided to carry out site-directed mutagenesis in this region to try to identify important amino acid residues for deoxysugar specificity in ElmGT.

A plasmid (pARGA) was constructed in which the elmGT and the elmE genes were cloned under the control of the ermE* promoter in plasmid pAR15AT (10). The elmE gene codes for a transmembrane protein responsible for secretion of elloramycin (20), and it was included in pARGA to facilitate secretion of the newly formed glycosylated derivatives. The targeted amino acids were located within a 344-bp FseI fragment internal to an 828-bp SalI-XhoI fragment. The latter fragment was rescued from pARGA and cloned for mutagenesis into pBluescript SK+ (Stratagene) to generate pSKGT0. Site-directed mutagenesis was carried out on this construct using the QuikChange II site-directed mutagenesis kit (Stratagene), and oligonucleotides designed to introduce specific changes in this amino acid region are shown in Table 1. After verification of the mutagenesis by DNA sequencing, the FseI fragment was cloned back into pARGA through the replacement of the nonmutagenized native FseI fragment by the mutagenized one. The newly generated plasmids were now independently introduced by conjugation from Escherichia coli into a panel of Streptomyces lividans strains, each harboring a plasmid directing the biosynthesis of a different L- or D-deoxysugar: L-rhamnose (pLN2), L-olivose (pLN2), and D-olivose (pLNR) (21); L-digitoxose (pLNBIV) (4); and D-boivinose (pMP1B*II) (19). The structures of these deoxysugars are shown in Fig. 2A. The resulting recombinant strains were then assayed for their capability to glycosylate 8DMTC. The strains were grown on small agar squares (2 by 2 cm) containing 1.5 ml of R5A solid medium plus 100 µg/ml 8DMTC for 7 days at 30°C. Then, the agar was extracted with ethyl acetate, and after evaporation of the solvent, the extracts were fractionated by high-performance liquid chromatography. The different glycosylated derivatives were identified by their retention times in comparison with those of pure samples used as standards (2). The amounts of each of these compounds were determined by integrating the peak areas.

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TABLE 1. Oligonucleotides used for site-directed mutagenesis of ElmGT

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FIG. 2. (A) Structures of the different deoxysugars used in this work. (B) Influence of amino acid changes in residues L309 and N312 of ElmGT on the efficiency of deoxysugar transfer to 8DMTC. The different recombinant strains harboring plasmids making L-rhamnose (pLN2), L-olivose (pLN2), D-olivose (pLNR), L-digitoxose (pLNBIV), or D-boivinose (pMP1B*II) were grown in triplicate on small agar squares (2 by 2 cm) containing 1.5 ml of R5A solid medium plus 100 µg/ml 8DMTC for 7 days at 30°C. Then, the agar was extracted with ethyl acetate, and after evaporation of the solvent, the extracts were fractionated by high-performance liquid chromatography. The different glycosylated derivatives were identified by their retention times in comparison with those of pure samples used as standards. The amounts of each of these compounds were determined by integrating the peak areas. AU, arbitrary units; WT, wild type.

Preliminary experiments showed that mutations affecting residues A308, A310, and D311 completely abolished sugar transfer, while those affecting L309 and N312 gave some functional GTFs. We therefore concentrated further analysis on changes in these two amino acids. Figure 2B and Table 2 show the results obtained in the experiments described below.

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TABLE 2. Amino acid substitutions performed in ElmGT GTF and their effect on deoxysugar transfer to 8DMTC

Substitution for the aliphatic amino acid L309 by either glutamine (Q), histidine (H), asparagine (N), or arginine (R)—all of them charged amino acids and thus representing drastic changes with respect to leucine—did not abolish sugar transfer; however, the efficiency of the transfer was reduced in a range between 30% and 80%. It is noticeable that arginine is naturally present in the equivalent position in two D-olivosyl GTFs, MtmGIV and CmmGIV (Fig. 1B), but substitution L309R in ElmGT is not sufficient to maintain the ability to transfer D-olivose. Replacement of L309 by neutral amino acids revealed changes in the transfer pattern of ElmGT. Methionine (M) is present in the equivalent position in two L-rhamnosyl GTFs, as StfG and AraGT (Fig. 1B). Substitution L309M in ElmGT reduced the capability to transfer D-olivose and L-digitoxose by about 75%, while it did not affect the ability to transfer L-olivose and L-rhamnose. Interestingly, the transfer of D-boivinose increased 1.5-fold. It has been shown that StfG, which also contains methionine in this position, is also able to efficiently transfer D-boivinose (17). The presence of isoleucine (I) greatly increased the transfer of L-rhamnose by about twofold, while incorporation of D-boivinose was not affected and transfer of other deoxysugars was reduced between 30% and 80%. When L309 was replaced by alanine (A) or valine (V), transfer of L-olivose increased four- and sixfold, respectively, and the incorporation of D-olivose remained unchanged, while the transfer of L-rhamnose, L-digitoxose, and D-boivinose was reduced. Interestingly, substitution for L309 by the aromatic and polar amino acid tyrosine (Y), which contains a bulkier side chain than leucine, enhanced L-olivose transfer by twofold without affecting D-olivose and D-boivinose incorporation. Interestingly, substitution L309Y caused a reduction in the transfer of L-rhamnose, in spite of the fact that tyrosine is present in another L-rhamnosyl GTF, SpnG (Fig. 1B).

Replacement of N312 by aspartate (D), alanine (A), or tyrosine (Y) greatly reduced the efficiency of deoxysugar attachment to 8DMTC. In the case of cysteine (C), deoxysugar transfer was also greatly reduced, with the exception of L-olivose, which remained unchanged. However, replacement by threonine (T), serine (S), or glutamine (Q) increased the transfer of L-olivose between two- and threefold, while it reduced the attachment of D-olivose, L-digitoxose, L-rhamnose, and D-boivinose. Only in substitution N312S did the ability of mutant ElmGT to transfer D-olivose and L-digitoxose remain unchanged. It is worth mentioning that substitution N312Q is naturally present in a number of GTFs independently of the deoxysugars they recognize (Fig. 1B).

The results in this paper clearly show that the promiscuous GTF activity of ElmGT can be modulated by specific amino acid substitutions in residues located in a short region within the /β/ motif. The flexibility of ElmGT for the attachment of different deoxysugars to 8DMTC has been widely reported previously (9, 17-19, 21). However, the efficiency of transfer varies depending upon the particular deoxysugar. By introducing specific changes in the donor binding site, these efficiencies can be improved. Thus, substitution for L309 or N312 by several amino acids increase the transfer of L-olivose (L309A, L309V, L309Y, N312T, N312Q, and N312S), while others increase the efficiency of D-boivinose (L309M) or L-rhamnose (L309I) incorporation. In particular, N312T and N312Q substitutions have been shown to be specific changes for the attachment of L-olivose since, in these mutants, incorporation of all other deoxysugars was greatly reduced.

In conclusion, we have shown that it is possible to tune the efficiency of donor selectivity in GTFs by replacing specific amino acids from the C terminus. Increasing experimental evidence shows that it is possible to alter the glycosylation pattern of bioactive natural products by combinatorial biosynthesis based on substrate flexibility of GTFs. However, sometimes one of the problems is the low efficiency of the process. Based on the results in this work, by making judicious and selected amino acid changes in the /β/ motif of GTFs, it could be possible to increase selectively the efficiency of transfer of a given deoxysugar and thus to facilitate the formation of novel active glycoconjugates. However, at the moment no correlation between amino acid changes and deoxysugars to be transferred can be drawn. Further experiments will be needed to clarify this aspect.

 
This research was supported by the Spanish Ministry of Science and Innovation (BFU2006-00404 to J.A.S. and BIO2005-04115 to C.M.), Red Temática de Investigación Cooperativa de Centros de Cáncer (Ministry of Health; ISCIII-RETIC RD06/0020/0026), and the EU FP6 (ActinoGen; Integrated project no. 005224). We thank Obra Social Cajastur for financial support to Carlos Olano.

 
* Corresponding author. Mailing address: Departamento de Biología Funcional e Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Universidad de Oviedo, 33006 Oviedo, Spain. Phone and fax: 34 985 103652. E-mail: jasalas{at}uniovi.es

Published ahead of print on 20 February 2009.

Present address: Instituto de Biotecnología de León (INBIOTEC), Parque Científico de León, 24006 León, Spain.

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Journal of Bacteriology, April 2009, p. 2871-2875, Vol. 191, No. 8
0021-9193/09/$08.00+0     doi:10.1128/JB.01747-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ramos, A. Articles by Salas, J. A. Search for Related Content PubMed PubMed Citation Articles by Ramos, A. Articles by Salas, J. A.