Abstract
DNA methylation and demethylation regulate many crucial biological processes in mammals and are linked to many diseases. Active DNA demethylation is believed to be catalyzed by TET proteins and a putative DNA decarboxylase that may share some similarities in sequence, structure and catalytic mechanism with isoorotate decarboxylase (IDCase) that catalyzes decarboxylation of 5caU to U in fungi. We report here the structures of wild-type and mutant IDCases from Cordyceps militaris and Metarhizium anisopliae in apo form or in complexes with 5caU, U, and an inhibitor 5-nitro-uracil. IDCases adopt a typical (β/α)8 barrel fold of the amidohydrolase superfamily and function as dimers. A Zn2+ is bound at the active site and coordinated by four strictly conserved residues, one Asp and three His. The substrate is recognized by several strictly conserved residues. The functional roles of the key residues at the active site are validated by mutagenesis and biochemical studies. Based on the structural and biochemical data, we present for the first time a novel catalytic mechanism of decarboxylation for IDCases, which might also apply to other members of the amidohydrolase superfamily. In addition, our biochemical data show that IDCases can catalyze decarboxylation of 5caC to C albeit with weak activity, which is the first in vitro evidence for direct decarboxylation of 5caC to C by an enzyme. These findings are valuable in the identification of potential DNA decarboxylase in mammals.
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Abbreviations
- IDCase:
-
isoorotate decarboxylase
- T7H:
-
thymine-7-hydroxylase
- ACMSD:
-
2-amino-3-carboxymuconate-6-semialdhyde decarboxylase
- 5caU:
-
5-carboxyl-uracil (isoorotate)
- 5hmU:
-
5-hydroxymethyl-uracil
- 5fU:
-
5-formyl-uracil
- 5niU:
-
5-nitro-uracil
- 2-thioIOA:
-
5-carboxyl-2-thio-uracil
- 5mC:
-
5-methyl-cytosine
- 5hmC:
-
5-hydroxymethyl-cytosine
- 5fC:
-
5-formyl-cytosine
- 5caC:
-
5-carboxyl-cytosine
- RMSD:
-
root-mean-square deviation
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Acknowledgements
We thank the staff members at Shanghai Synchrotron Radiation Facility (SSRF) of China for technical supports in diffraction data collection, and other members of our groups for discussion. This work was supported by grants from the Ministry of Science and Technology of China (2011CB966301), the National Natural Science Foundation of China (31221001), and the Science and Technology Commission of Shanghai Municipality (10JC1416500).
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1
The chemistry for the conversion of 5mC to C in mammals is very similar to that for the conversion of T to U in the thymidine salvage pathway in fungi. (PDF 151 kb)
Supplementary information, Figure S2
Topology of the overall structural fold of CmIDCase. (PDF 76 kb)
Supplementary information, Figure S3
Comparison of IDCases from different fungi species. (PDF 1485 kb)
Supplementary information, Figure S4
CmIDCase and MaIDCase exist as dimers in both solution and crystal structure. (PDF 132 kb)
Supplementary information, Figure S5
Anomalous dispersion analyses of CmIDCase and MaIDCase. (PDF 54 kb)
Supplementary information, Figure S6
Structure of the active site. (PDF 1417 kb)
Supplementary information, Figure S7
Kinetic assays of the decarboxylation activities of wild-type CmIDCase and MaIDCase. (PDF 49 kb)
Supplementary information, Figure S8
Comparison of CmIDCase with members of the (α/β)8 barrel-containing amidohydrolase superfamily. (PDF 1487 kb)
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Xu, S., Li, W., Zhu, J. et al. Crystal structures of isoorotate decarboxylases reveal a novel catalytic mechanism of 5-carboxyl-uracil decarboxylation and shed light on the search for DNA decarboxylase. Cell Res 23, 1296–1309 (2013). https://doi.org/10.1038/cr.2013.107
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DOI: https://doi.org/10.1038/cr.2013.107
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