Characterization of the Calicheamicin Orsellinate C2‐<i>O</i>‐Methyltransferase CalO6

Singh, Shanteri; Nandurkar, Nitin S.; Thorson, Jon S.

doi:10.1002/cbic.201402119

Cited by 10 publications

(12 citation statements)

References 26 publications

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“…Genomic analysis, gene disruption, and structural characterization with ligands can suggest function for natural product biosynthetic enzymes (15,38,39), and our data strongly support a role for HygX in catalyzing orthoester linkage formation. Together, these data suggest a fully decorated three-ringed compound lacking the orthoester linkage as the substrate and narrows the possibilities for HygX substrates to two alternative anomers at C1 of the destomic acid moiety.…”

Section: Discussionsupporting

confidence: 61%

Oxidative cyclizations in orthosomycin biosynthesis expand the known chemistry of an oxygenase superfamily

McCulloch

McCranie

Smith

et al. 2015

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

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Orthosomycins are oligosaccharide antibiotics that include avilamycin, everninomicin, and hygromycin B and are hallmarked by a rigidifying interglycosidic spirocyclic ortho-δ-lactone (orthoester) linkage between at least one pair of carbohydrates. A subset of orthosomycins additionally contain a carbohydrate capped by a methylenedioxy bridge. The orthoester linkage is necessary for antibiotic activity but rarely observed in natural products. Orthoester linkage and methylenedioxy bridge biosynthesis require similar oxidative cyclizations adjacent to a sugar ring. We have identified a conserved group of nonheme iron, α-ketoglutarate-dependent oxygenases likely responsible for this chemistry. High-resolution crystal structures of the EvdO1 and EvdO2 oxygenases of everninomicin biosynthesis, the AviO1 oxygenase of avilamycin biosynthesis, and HygX of hygromycin B biosynthesis show how these enzymes accommodate large substrates, a challenge that requires a variation in metal coordination in HygX. Excitingly, the ternary complex of HygX with cosubstrate α-ketoglutarate and putative product hygromycin B identified an orientation of one glycosidic linkage of hygromycin B consistent with metal-catalyzed hydrogen atom abstraction from substrate. These structural results are complemented by gene disruption of the oxygenases evdO1 and evdMO1 from the everninomicin biosynthetic cluster, which demonstrate that functional oxygenase activity is critical for antibiotic production. Our data therefore support a role for these enzymes in the production of key features of the orthosomycin antibiotics.antibiotic biosynthesis | oxidative cyclization | crystal structure | nonheme iron α-ketoglutarate-dependent oxygenases

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Section: Discussionsupporting

confidence: 61%

Oxidative cyclizations in orthosomycin biosynthesis expand the known chemistry of an oxygenase superfamily

McCulloch

McCranie

Smith

et al. 2015

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

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“…Cell disruption was achieved by sonication on ice in pH 8.8 reaction buffer (40 mM Tris HCl, 100 mM NaCl). Initial bioconversion experiments were carried out with the racemic mixture of substrate surrogates rac - 14d (0.25 mM), 5 mM MgCl 2 and 0.97 mM SAM tosylate for 16 h at 28 °C (Figure 1, for unprocessed spectra see Figures S48–S53) [26,27]. HPLC-MS analysis showed that rac - 14d was almost completely converted into the respective O -methylated product mixture rac - 15d (Figure 1f).…”

Section: Resultsmentioning

confidence: 99%

Characterisation of the Broadly-Specific O-Methyl-transferase JerF from the Late Stages of Jerangolid Biosynthesis

et al. 2016

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Abstract:We describe the characterisation of the O-methyltransferase JerF from the late stages of jerangolid biosynthesis. JerF is the first known example of an enzyme that catalyses the formation of a non-aromatic, cyclic methylenolether. The enzyme was overexpressed in E. coli and the cell-free extracts were used in bioconversion experiments. Chemical synthesis gave access to a series of substrate surrogates that covered a broad structural space. Enzymatic assays revealed a broad substrate tolerance and high regioselectivity of JerF, which makes it an attractive candidate for an application in chemoenzymatic synthesis with particular usefulness for late stage application on 4-methoxy-5,6-dihydro-2H-pyran-2-one-containing natural products.

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“…Likewise, CalO6 did not form suitable crystals in the presence of 1-2 mM SAM, SAH, or 2-5 mM substrate N -acetylcysteamine orsellinic acid (SNAC-OSA; used with or without SAH), nor did we observe these ligands in the electron density map upon soaking them into crystals of CalO6 grown in their absence. These ligand concentrations were several-fold higher than the previously reported K m values (0.3 mM for SAM and 1.3 mM for SNAC-OSA) [ 8 ], which ensured that most CalO6 is in a ligand-bound form at the conditions of the reported activity assays. However, we could not exclude a possibility that the ligand binding was disfavored in the crystallization solution.…”

Section: Methodsmentioning

confidence: 75%

“…Fractions containing pure CalO6 (as determined by SDS-PAGE), were pooled and the protein was concentrated to ~14 mg/mL in an Amicon Ultra-15 filtration unit and stored at 4 °C for use in crystallization experiments. The purified protein (Additional file 1 : Figure S1 in Supporting information) was assayed as reported previously [ 8 ] and exhibited similar activity in methylating S - N -acetylcysteaminyl orsellinic acid, as detected by HPLC. SeMet-substituted CalO6 was prepared as previously described [ 11 ] and purified analogously to the unsubstituted CalO6.…”

Section: Methodsmentioning

confidence: 99%

“…A recent report suggests that the 2-hydroxyl group methylation is carried out by CalO6 as the first tailoring reaction of the orsellinic acid moiety likely attached to the acyl carrier protein (ACP) domain of CalO5 (indicated by R in Fig. 1 ) [ 8 ], whereas another candidate O -methyltransferase, CalO1, is inert when tested on the same substrate analogue [ 9 ].

Fig.…”

Section: Introductionmentioning

confidence: 99%

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Crystal structure of O-methyltransferase CalO6 from the calicheamicin biosynthetic pathway: a case of challenging structure determination at low resolution

2015

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BackgroundCalicheamicins (CAL) are enedyine natural products with potent antibiotic and cytotoxic activity, used in anticancer therapy. The O-methyltransferase CalO6 is proposed to catalyze methylation of the hydroxyl moiety at the C2 position of the orsellinic acid group of CAL.ResultsCrystals of CalO6 diffracted non-isotropically, with the usable data extending to 3.4 Å. While no single method of crystal structure determination yielded a structure of CalO6, we were able to determine its structure by using molecular replacement-guided single wavelength anomalous dispersion by using diffraction data from native crystals of CalO6 and a highly non-isomorphous mercury derivative. The structure of CalO6 reveals the methyltransferase fold and dimeric organization characteristic of small molecule O-methyltransferases involved in secondary metabolism in bacteria and plants. Uncommonly, CalO6 was crystallized in the absence of S-adenosylmethionine (SAM; the methyl donor) or S-adenosylhomocysteine (SAH; its product).ConclusionsLikely as a consequence of the dynamic nature of CalO6 in the absence of its cofactor, the central region of CalO6, which forms a helical lid-like structure near the active site in CalO6 and similar enzymes, is not observed in the electron density. We propose that this region controls the entry of SAM into and the exit of SAH from the active site of CalO6 and shapes the active site for substrate binding and catalysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12900-015-0040-6) contains supplementary material, which is available to authorized users.

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Characterization of the Calicheamicin Orsellinate C2‐O‐Methyltransferase CalO6

Cited by 10 publications

References 26 publications

Oxidative cyclizations in orthosomycin biosynthesis expand the known chemistry of an oxygenase superfamily

Oxidative cyclizations in orthosomycin biosynthesis expand the known chemistry of an oxygenase superfamily

Characterisation of the Broadly-Specific O-Methyl-transferase JerF from the Late Stages of Jerangolid Biosynthesis

Crystal structure of O-methyltransferase CalO6 from the calicheamicin biosynthetic pathway: a case of challenging structure determination at low resolution

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