Biochemical and Structural Insights of the Early Glycosylation Steps in Calicheamicin Biosynthesis

Bitto, E.; Goff, Randal D.; Singh, Shanteri; Bingman, C.A.; Griffith, Byron R.; Albermann, Christoph; Phillips, George N.; Thorson, Jon S.

doi:10.1016/j.chembiol.2008.06.011

Cited by 50 publications

(56 citation statements)

References 70 publications

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“…CLM α 3 I was provided by Pfizer. CLM T 0 was prepared as previously described (16). CalG3, CalG2, and CalG1 with TDP samples were prepared by mixing 10 mg∕mL of CalG3 or 20 mg∕mL of CalG2 or CalG1 protein samples with 25 mM TDP.…”

Section: Methodsmentioning

confidence: 99%

“…1). Some CLM GTs are highly promiscuous and can perform forward, reverse, and exchange reactions, enabling chemoenzymatic methods to generate glycodiversified CLM analogs (15,16). Based upon biochemical studies, CalG1 and CalG4 were found to be external GTs, acting as a rhamnosyltransferase for sugar moiety D and as an aminopentosyltransferase for sugar moiety E, respectively.…”

mentioning

confidence: 99%

“…1). Previously, a CalG3 unliganded structure was reported (16); however, the absence of substrates in the model prevented understanding of the binding mode of CLM and identification of the origins of regiospecificity.…”

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confidence: 99%

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Complete set of glycosyltransferase structures in the calicheamicin biosynthetic pathway reveals the origin of regiospecificity

Chang

Singh

Helmich

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Glycosyltransferases are useful synthetic catalysts for generating natural products with sugar moieties. Although several natural product glycosyltransferase structures have been reported, design principles of glycosyltransferase engineering for the generation of glycodiversified natural products has fallen short of its promise, partly due to a lack of understanding of the relationship between structure and function. Here, we report structures of all four calicheamicin glycosyltransferases (CalG1, CalG2, CalG3, and CalG4), whose catalytic functions are clearly regiospecific. Comparison of these four structures reveals a conserved sugar donor binding motif and the principles of acceptor binding region reshaping. Among them, CalG2 possesses a unique catalytic motif for glycosylation of hydroxylamine. Multiple glycosyltransferase structures in a single natural product biosynthetic pathway are a valuable resource for understanding regiospecific reactions and substrate selectivities and will help future glycosyltransferase engineering.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Complete set of glycosyltransferase structures in the calicheamicin biosynthetic pathway reveals the origin of regiospecificity

Chang

Singh

Helmich

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Cpz31, together with most glycosyltransferases involved in natural product biosynthesis, belongs to the GT-1 family of glycosyltransferases as classified by the CAZy system (6). Cpz31 exhibits significantly higher homology to, for example, the 4,6-dideoxy-4-hydroxylamino-␣-Dglucosyltransferase CalG3 (36% identity, 50% similarity) from Micromonospora echinospora (59) than to the rhamnosyltransferases AraGT (29% identiy, 38% similarity) from S. echinatus (38) and SpnG (29% identity, 39% similarity) from Saccharopolyspora spinosa (5). Luzhetskyy et al (39) proposed that similarity-based relationships of glycosyltransferases are better understood with consideration of the aglycone substrate than of the sugar donor.…”

Section: Discussionmentioning

confidence: 99%

Formation and Attachment of the Deoxysugar Moiety and Assembly of the Gene Cluster for Caprazamycin Biosynthesis

Kaysser

Wemakor

Siebenberg

et al. 2010

Appl Environ Microbiol

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Caprazamycins are antimycobacterials produced by Streptomyces sp. MK730-62F2. Previously, cosmid cpzLK09 was shown to direct the biosynthesis of caprazamycin aglycones, but not of intact caprazamycins. Sequence analysis of cpzLK09 identified 23 genes involved in the formation of the caprazamycin aglycones and the transfer and methylation of the sugar moiety, together with genes for resistance, transport, and regulation. In this study, coexpression of cpzLK09 in Streptomyces coelicolor M512 with pRHAM, containing all the required genes for dTDP-L-rhamnose biosynthesis, led to the production of intact caprazamycins. In vitro studies showed that Cpz31 is responsible for the attachment of the L-rhamnose to the caprazamycin aglycones, generating a rare acylated deoxyhexose. An L-rhamnose gene cluster was identified elsewhere on the Streptomyces sp. MK730-62F2 genome, and its involvement in caprazamycin formation was demonstrated by insertional inactivation of cpzDIII. The L-rhamnose subcluster was assembled with cpzLK09 using Red/ET-mediated recombination. Heterologous expression of the resulting cosmid, cpzEW07, led to the production of caprazamycins, demonstrating that both sets of genes are required for caprazamycin biosynthesis. Knockouts of cpzDI and cpzDV in the L-rhamnose subcluster confirmed that four genes, cpzDII, cpzDIII, cpzDIV, and cpzDVI, are sufficient for the biosynthesis of the deoxysugar moiety. The presented recombineering strategy may provide a useful tool for the assembly of biosynthetic building blocks for heterologous production of microbial compounds.Caprazamycins are potent antimycobacterials isolated from Streptomyces sp. MK730F-62F2 (23). In a pulmonary tuberculosis mouse model, they showed a therapeutic effect but no significant toxicity (24). The caprazamycins are assigned to the translocase I inhibitors (27) due to their structural similarity to the liposidomycins (34, 43), which have been studied in more detail. Translocase I catalyzes the first step in the membranelinked reaction cycle of bacterial cell wall formation (52): the transfer of phospho-N-acetylmuramic acid-L-Ala-␥-D-Glu-mdiaminopimelic acid-D-Ala-D-Ala from UMP to the lipid carrier undecaprenyl phosphate. Structurally unique in nature, the caprazamycins and liposidomycins share a 5Ј-␤-O-aminoribosyl-glycyluridine and a rare N-methylated diazepanone as their characteristic feature (Fig. 1) (25, 53). Attached at the 3Љ position are ␤-hydroxylated fatty acid groups of different chain lengths, carrying a 3-methylglutarate. While the liposidomycins are sulfated, the caprazamycins lack this group. Instead, they are glycosylated with a 2,3,4-O-methyl-L-rhamnose and therefore belong to the large number of bioactive compounds containing 6-deoxyhexoses. Usually, these moieties contribute significantly to the compounds' properties, influencing, e.g., molecule-target interactions, cell import and export, pharmacokinetics, and solubility (56). The biosynthesis of deoxysugars has been studied in detail and generally starts from NDPactiva...

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“…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)(15)(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).…”

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confidence: 99%

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

et al. 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 ␣/␤/␣ 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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Biochemical and Structural Insights of the Early Glycosylation Steps in Calicheamicin Biosynthesis

Cited by 50 publications

References 70 publications

Complete set of glycosyltransferase structures in the calicheamicin biosynthetic pathway reveals the origin of regiospecificity

Complete set of glycosyltransferase structures in the calicheamicin biosynthetic pathway reveals the origin of regiospecificity

Formation and Attachment of the Deoxysugar Moiety and Assembly of the Gene Cluster for Caprazamycin Biosynthesis

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

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