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Crystal structure of O-methyltransferase CalO6 from the calicheamicin biosynthetic pathway: a case of challenging
Oleg V Tsodikov1, Caixia Hou2, Christopher T Walsh3
1Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, 40536-0596, Lexington, KY, USA. oleg.tsodikov@uky.edu.
Background:
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.
Results:
Crystals 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).
Conclusions:
Likely 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.
Insights
The O-methyltransferase CalO6, involved in calicheamicin biosynthesis, was structurally determined using anomalous dispersion. Its structure reveals a typical methyltransferase fold, but a key active site lid is disordered in the absence of cofactors.
Area of Science:
- Biochemistry
- Structural Biology
- Natural Product Biosynthesis
Background:
- Calicheamicins (CAL) are potent enediyne natural products with antibiotic and cytotoxic activities, utilized in anticancer therapy.
- The O-methyltransferase CalO6 is implicated in catalyzing the methylation of the orsellinic acid moiety in CAL biosynthesis.
Purpose of the Study:
- To determine the crystal structure of the O-methyltransferase CalO6.
- To elucidate the structural features of CalO6 and its potential role in calicheamicin biosynthesis.
Main Methods:
- Crystallization of CalO6.
- Molecular replacement-guided single wavelength anomalous dispersion (SAD) phasing.
- X-ray diffraction data collection and structure determination.
Main Results:
- The crystal structure of CalO6 was determined to 3.4 Å resolution using molecular replacement-guided SAD.
- CalO6 exhibits the characteristic methyltransferase fold and dimeric organization.
- Crystallization occurred in the absence of the methyl donor S-adenosylmethionine (SAM) or its product S-adenosylhomocysteine (SAH), leading to disorder in a key active site lid region.
Conclusions:
- The observed disorder of the active site lid in CalO6, likely due to the absence of cofactors, suggests its dynamic nature.
- This lid region is proposed to regulate the binding and release of SAM and SAH.
- The lid is hypothesized to control substrate access and shape the active site for catalysis in CalO6.
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