Abstract
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.
Highlights
Calicheamicins (CAL) are enedyine natural products with potent antibiotic and cytotoxic activity, used in anticancer therapy
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 [8], whereas another candidate O-methyltransferase, CalO1, is inert when tested on the same substrate analogue [9]
CalO6 is a rare O-methyltransferase crystallized in the absence of cofactors or substrates; co-crystals with its ligands either did not grow or were not of high enough quality for data collection, and crystal soaking experiments did not yield stably bound complexes, likely because those were disfavored by the crystallization conditions or crystal packing
Summary
Calicheamicins (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. Calicheamicins (CAL) are bacterial natural products with antibiotic and antitumor activities, which originate from the ability of these compounds to cleave double-stranded DNA [1]. Protein CalO6 from the CAL gene cluster was originally proposed to catalyze one or both O-methylations (at positions C2 and/or C3) of this moiety [7]. 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]
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