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Characterization of the calicheamicin orsellinate C2-O-methyltransferase CalO6.

ABSTRACT: Although bacterial iterative type I polyketide synthases are now known to participate in the biosynthesis of a small set of diverse natural products, the subsequent downstream modification of the resulting polyketide products is poorly understood. We report the functional characterization of the putative orsellinic acid C2-O-methyltransferase, which is involved in calicheamicin biosynthesis. This study suggests that C2-O-methylation precedes C3-hydroxylation/methylation and C5-iodination and requires a coenzyme A- or acyl carrier protein-bound substrate.

SUBMITTER: Singh S

PROVIDER: S-EPMC4111469 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Characterization of the calicheamicin orsellinate C2-O-methyltransferase CalO6.

Singh Shanteri S Nandurkar Nitin S NS Thorson Jon S JS

Chembiochem : a European journal of chemical biology 20140701 10

Although bacterial iterative type I polyketide synthases are now known to participate in the biosynthesis of a small set of diverse natural products, the subsequent downstream modification of the resulting polyketide products is poorly understood. We report the functional characterization of the putative orsellinic acid C2-O-methyltransferase, which is involved in calicheamicin biosynthesis. This study suggests that C2-O-methylation precedes C3-hydroxylation/methylation and C5-iodination and req ...[more]

PMID: 24978950

Similar Datasets

Structural characterization of CalO1: a putative orsellinic acid methyltransferase in the calicheamicin-biosynthetic pathway.

Project description:The X-ray structure determination at 2.4?Å resolution of the putative orsellinic acid C3 O-methyltransferase (CalO1) involved in calicheamicin biosynthesis is reported. Comparison of CalO1 with a homology model of the functionally related calicheamicin orsellinic acid C2 O-methyltransferase (CalO6) implicates several residues that are likely to contribute to the regiospecificity of alkylation. Consistent with the proposed requirement of an acyl-carrier-protein-bound substrate, this structural study also reveals structural determinants within CalO1 that are anticipated to accommodate an association with an acyl carrier protein.

| S-EPMC3046457 | biostudies-literature

Structural and functional characterization of CalS11, a TDP-rhamnose 3'-O-methyltransferase involved in calicheamicin biosynthesis.

Project description:Sugar methyltransferases (MTs) are an important class of tailoring enzymes that catalyze the transfer of a methyl group from S-adenosyl-l-methionine to sugar-based N-, C- and O-nucleophiles. While sugar N- and C-MTs involved in natural product biosynthesis have been found to act on sugar nucleotide substrates prior to a subsequent glycosyltransferase reaction, corresponding sugar O-methylation reactions studied thus far occur after the glycosyltransfer reaction. Herein we report the first in vitro characterization using (1)H-(13)C-gHSQC with isotopically labeled substrates and the X-ray structure determination at 1.55 Å resolution of the TDP-3'-O-rhamnose-methyltransferase CalS11 from Micromonospora echinospora. This study highlights a unique NMR-based methyltransferase assay, implicates CalS11 to be a metal- and general acid/base-dependent O-methyltransferase, and as a first crystal structure for a TDP-hexose-O-methyltransferase, presents a new template for mechanistic studies and/or engineering.

| S-EPMC3875630 | biostudies-literature

Loop dynamics of thymidine diphosphate-rhamnose 3'-O-methyltransferase (CalS11), an enzyme in calicheamicin biosynthesis.

Project description:Structure analysis and ensemble refinement of the apo-structure of thymidine diphosphate (TDP)-rhamnose 3'-O-methyltransferase reveal a gate for substrate entry and product release. TDP-rhamnose 3'-O-methyltransferase (CalS11) catalyses a 3'-O-methylation of TDP-rhamnose, an intermediate in the biosynthesis of enediyne antitumor antibiotic calicheamicin. CalS11 operates at the sugar nucleotide stage prior to glycosylation step. Here, we present the crystal structure of the apo form of CalS11 at 1.89?Å resolution. We propose that the L2 loop functions as a gate facilitating and/or providing specificity for substrate entry or promoting product release. Ensemble refinement analysis slightly improves the crystallographic refinement statistics and furthermore provides a compelling way to visualize the dynamic model of loop L2, supporting the understanding of its proposed role in catalysis.

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Characterization of CalE10, the N-oxidase involved in calicheamicin hydroxyaminosugar formation.

Project description:As the first in vitro characterization of a sugar N-oxidase, this study establishes CalE10 as the key oxidase involved in calicheamicin hydroxylamino glycoside formation. This study confirms that oxidation occurs at the sugar nucleotide stage prior to glycosyltransfer, and substrate specificity studies reveal CalE10-catalyzed oxidation to be regiospecific and to present trace amounts of the corresponding nitrosugar in vitro. This work also sets a precedent for the future study of other N-oxidases involved in hydroxylamino-, nitroso-, and/or nitrosugar formation.

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

Project description: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.

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Structural Characterization of CalS8, a TDP-α-D-Glucose Dehydrogenase Involved in Calicheamicin Aminodideoxypentose Biosynthesis.

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Structural characterization of CalO2: a putative orsellinic acid P450 oxidase in the calicheamicin biosynthetic pathway.

Project description:Although bacterial iterative Type I polyketide synthases are now known to participate in the biosynthesis of a small set of diverse natural products, the subsequent downstream modification of the resulting polyketide products remains poorly understood. Toward this goal, we report the X-ray structure determination at 2.5 A resolution and preliminary characterization of the putative orsellenic acid P450 oxidase (CalO2) involved in calicheamicin biosynthesis. These studies represent the first crystal structure for a P450 involved in modifying a bacterial iterative Type I polyketide product and suggest the CalO2-catalyzed step may occur after CalO3-catalyzed iodination and may also require a coenzyme A- (CoA) or acyl carrier protein- (ACP) bound substrate. Docking studies also reveal a putative docking site within CalO2 for the CLM orsellinic acid synthase (CalO5) ACP domain which involves a well-ordered helix along the CalO2 active site cavity that is unique compared with other P450 structures.

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Glycine N-methyltransferase deficiency affects Niemann-Pick type C2 protein stability and regulates hepatic cholesterol homeostasis.

Project description:Nonalcoholic fatty liver disease (NAFLD) is associated with the development of metabolic syndromes and hepatocellular carcinoma (HCC). Cholesterol accumulation is related to NAFLD, whereas its detailed mechanism is not fully understood. Previously, we reported that glycine N-methyltransferase (GNMT) knockout (Gnmt(-/-)) mice develop chronic hepatitis and HCC. In this study, we showed that Gnmt(-/-) mice had hyperlipidemia and steatohepatitis. Single photon emission computed tomography images of mice injected with (131)I-labeled 6?-iodocholesterol demonstrated that Gnmt(-/-) mice had slower hepatic cholesterol uptake and excretion rates than wild-type mice. In addition, genes related to cholesterol uptake (scavenger receptor class B type 1 [SR-B1] and ATP-binding cassette A1 [ABCA1]), intracellular trafficking (Niemann-Pick type C1 protein [NPC1] and Niemann-Pick type C2 protein [NPC2]) and excretion (ATP-binding cassette G1 [ABCG1]) were downregulated in Gnmt(-/-) mice. Yeast two-hybrid screenings and coimmunoprecipitation assays elucidated that the C conserved region (81-105 amino acids) of NPC2 interacts with the carboxyl-terminal fragment (171-295 amino acids) of GNMT. Confocal microscopy demonstrated that when cells were treated with low-density lipoprotein, NPC2 was released from lysosomes and interacts with GNMT in the cytosol. Overexpression of GNMT doubled the half-lives of both NPC2 isoforms and reduced cholesterol accumulation in cells. Furthermore, GNMT was downregulated in the liver tissues from patients suffering with NAFLD as well as from mice fed a high-fat diet, high-cholesterol diet or methionine/choline-deficient diet. In conclusion, our study demonstrated that GNMT regulates the homeostasis of cholesterol metabolism, and hepatic cholesterol accumulation may result from downregulation of GNMT and instability of its interactive protein NPC2. Novel therapeutics for steatohepatitis and HCC may be developed by using this concept.

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Characterization of SETD3 methyltransferase-mediated protein methionine methylation.

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| S-EPMC7415985 | biostudies-literature

Structural characterization of the mitomycin 7-O-methyltransferase.

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