Iteratively Acting Glycosyltransferases Involved in the Hexasaccharide Biosynthesis of Landomycin A

Luzhetskyy, Andriy; Fedoryshyn, Marta; Dürr, Clemens; Taguchi, Tanezo; Novikov, Volodymyr; Bechthold, Andreas

doi:10.1016/j.chembiol.2005.05.008

Cited by 57 publications

(65 citation statements)

References 17 publications

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“…Iterative use of glycosltransferases, however, is not unprecedented in natural product biosynthesis. In landomycin A biosynthesis, LanGT1 (an olivosyltransferase) and LanGT4 (an rhodinosyltransferase) act twice, with each transferring two of the sugars (olivose and rhodinose) found in the hexasaccharide portion of the landomycin A structure (24). Ramoplanin, like mannopeptimycin, contains an O-linked di-mannose, and in the ramoplanin gene cluster there is only one glycosyltransferase (Ram29) (11).…”

Section: Resultsmentioning

confidence: 99%

Biosynthetic Pathway for Mannopeptimycins, Lipoglycopeptide Antibiotics Active against Drug-Resistant Gram-Positive Pathogens

Magarvey¹,

Haltli²,

He³

et al. 2006

Antimicrob Agents Chemother

108

116

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The mannopeptimycins are a novel class of lipoglycopeptide antibiotics active against multidrug-resistant pathogens with potential as clinically useful antibacterials. This report is the first to describe the biosynthesis of this novel class of mannosylated lipoglycopeptides. Included here are the cloning, sequencing, annotation, and manipulation of the mannopeptimycin biosynthetic gene cluster from Streptomyces hygroscopicus NRRL 30439. Encoded by genes within the mannopeptimycin biosynthetic gene cluster are enzymes responsible for the generation of the hexapeptide core (nonribosomal peptide synthetases [NRPS]) and tailoring reactions (mannosylation, isovalerylation, hydroxylation, and methylation). The NRPS system is noncanonical in that it has six modules utilizing only five amino acid-specific adenylation domains and it lacks a prototypical NRPS macrocyclizing thioesterase domain. Analysis of the mannopeptimycin gene cluster and its engineering has elucidated the mannopeptimycin biosynthetic pathway and provides the framework to make new and improved mannopeptimycins biosynthetically.Multidrug-resistant gram-positive bacterial pathogens now exist, and their rising numbers are a major concern. The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) exemplifies the seriousness of this concern (22, 29). Finding novel antibiotics active against such drug-resistant gram-positive pathogens, however, is problematic (32, 42). The mannopeptimycins ( Fig. 1), which are produced by Streptomyces hygroscopicus NRRL 30439, represent a new class of lipoglycopeptide antibiotics with exceptional in vitro and in vivo antibacterial activities against MRSA, VRE, and penicillin-resistant Streptococcus pneumoniae (17,36). Studies with mannopeptimycin-␦ demonstrated that this antibiotic acts by blocking the transglycosylation reaction in cell wall biosynthesis mediated by lipid II binding. Mannopeptimycin-␦ inhibits gram-positive cell wall biosynthesis through a mechanism which does not compete or render it ineffective due to crossresistance with the vancomycin-type antibiotics (33, 36).The unique structure, mode of action, and bioactivity of the mannopeptimycins have generated much interest in identifying a therapeutic candidate from this new class of molecules (17,33,36). One semisynthetic analog, AC98-6446, of the natural mannopeptimycins is significantly more potent and effective than the natural mannopeptimycins, with MICs in the 15-to 60-ng/ml range against MRSA, VRE, penicillin-resistant Streptococcus pneumoniae, and glycopeptide-intermediate Staphylococcus aureus (10, 31). The success in improving on the natural mannopeptimycins argues strongly for broadening the effort of generating additional new mannopeptimycins through promising strategies such as combinatorial biosynthesis, mutasynthesis, and chemoenzymatic synthesis (4, 28). The establishment and success of such approaches hinge on the cloning of the mannopeptimycin biosynthetic gene cluster and elucidation of ...

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Section: Resultsmentioning

confidence: 99%

Biosynthetic Pathway for Mannopeptimycins, Lipoglycopeptide Antibiotics Active against Drug-Resistant Gram-Positive Pathogens

Magarvey¹,

Haltli²,

He³

et al. 2006

Antimicrob Agents Chemother

108

116

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“…While LandGT1 is responsible for the transfer of the second and fifth sugar moiety, LanGT4 transfers both rhodinoses into the third and sixth positions. [16] Most recently, LanGT2 was identified to catalyze the first glycosylation step (D-olivosyl), [17] and a targeted gene inactivation experiment on lanGT3 showed that the corresponding glycosyltransferase LanGT3 is an olivosyltransferase. [18] LanGT3 is most likely responsible for the attachment of the fourth sugar, which is the key step that distinguishes the biosyntheses of LaA and LaE.…”

Section: Introductionmentioning

confidence: 99%

Generation of New Landomycins with Altered Saccharide Patterns through Over‐expression of the Glycosyltransferase Gene lanGT3 in the Biosynthetic Gene Cluster of Landomycin A in Streptomyces cyanogenus S‐136

et al. 2006

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Two novel landomycin compounds, landomycins I and J, were generated with a new mutant strain of Streptomyces cyanogenus in which the glycosyltransferase that is encoded by lanGT3 was overexpressed. This mutant also produced the known landomycins A, B, and D. All these compounds consist ofthe same polyketide-derived aglycon but differ in their sugar moieties, which are chains of different lengths. The major new metabolite, landomycin J, was found to consist of landomycinone with a tetrasaccharide chain attached. Combined with previous results ofthe production oflandomycin E (which contains three sugars) by the LanGT3 -mutant strain (obtained by targeted gene deletion of lanGT3), it was verified that LanGT3 is a D-olivosyltransferase responsible for the transfer of the fourth sugar required for landomycin A biosynthesis. The experiments also showed that gene overexpression is a powerful method for unbalancing biosynthetic pathways in order to generate new metabolites. The cytotoxicity ofthe new landomycins-compared to known ones-was assessed by using three different tumor cell lines, and their structure-activity relationship (SAR) with respect to the length ofthe deoxysugar side chain was deduced from the results.

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“…PnxGT2 belongs to glycosyltransferase family 28 and shows similarity to many O-glycosyltransferases, including LanGT1 (62%), LanGT3 (55%) and LanGT4 (47%) in landomycin biosynthesis and AtG1 (50%) in AT2433 biosynthesis. Because LanGT1 and LanGT4 were confirmed to be used iteratively in landomycin biosynthesis, 44 PnxGT2 was proposed to similarly catalyze two glycosylations to complete the trisaccharide formation in FD-594 biosynthesis.…”

Section: Glycosyltransferases and Methyltransferasesmentioning

confidence: 99%

Cloning of the biosynthetic gene cluster for naphthoxanthene antibiotic FD-594 from Streptomyces sp. TA-0256

et al. 2010

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FD-594 is an unique pyrano [4¢,3¢:6,7]naphtho [1,2-b]xanthene polyketide with a trisaccharide of 2,6-dideoxysugars. In this study, we cloned the FD-594 biosynthetic gene cluster from the producer strain Streptomyces sp. TA-0256 to investigate its biosynthesis. The identified pnx gene cluster was 38 143 bp, consisting of 40 open reading frames, including a minimal PKS gene, TDP-olivose biosynthetic genes, two glycosyltransferase genes, two methyltransferase genes and many oxygenase/reductase genes. Most of these enzymes coded in the pnx cluster were reasonably assigned to a plausible biosynthetic pathway for FD-594, in which an unique ring opening process via Baeyer-Villiger-type oxidation catalyzed by a putative flavin adenine dinucleotide (FAD)-dependent monooxygenase, is speculated to lead to the unique xanthene structure. To clarify the involvement of pnx genes in the FD-594 biosynthesis, a glycosyltransferase, PnxGT2, and a methyltransferase, PnxMT2, were characterized enzymatically with the recombinant proteins expressed in Escherichia coli. As a result, PnxGT2 catalyzed the triple olivose transfers to the FD-594 aglycon with TDP-olivose as the glycosyl donor to afford triolivoside. Surprisingly, in the PnxGT2 enzymatic reaction, tetraolivoside and pentaolivoside were significantly detected along with the expected triolivoside. To our knowledge, PnxGT2 is the first contiguous oligosaccharide-forming glycosyltransferase in secondary metabolism. Furthermore, addition of PnxMT2 and S-adenosyl-L-methionine into the PnxGT2 reaction mixture afforded natural FD-594 to confirm that the PnxGT2 reaction product was the expected regiospecifically glycosylated compound. Consequently, the identified pnx gene cluster appears to be involved in FD-594 biosynthesis.

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Iteratively Acting Glycosyltransferases Involved in the Hexasaccharide Biosynthesis of Landomycin A

Cited by 57 publications

References 17 publications

Biosynthetic Pathway for Mannopeptimycins, Lipoglycopeptide Antibiotics Active against Drug-Resistant Gram-Positive Pathogens

Biosynthetic Pathway for Mannopeptimycins, Lipoglycopeptide Antibiotics Active against Drug-Resistant Gram-Positive Pathogens

Generation of New Landomycins with Altered Saccharide Patterns through Over‐expression of the Glycosyltransferase Gene lanGT3 in the Biosynthetic Gene Cluster of Landomycin A in Streptomyces cyanogenus S‐136

Cloning of the biosynthetic gene cluster for naphthoxanthene antibiotic FD-594 from Streptomyces sp. TA-0256

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