Research Article: Purification, crystallization and X-ray structures of the two manganese superoxide dismutases from Caenorhabditis elegans

Date Published: December 1, 2008

Publisher: International Union of Crystallography

Author(s): Chi H. Trinh, Thérèse Hunter, Emma E. Stewart, Simon E. V. Phillips, Gary J. Hunter.

http://doi.org/10.1107/S1744309108037056

Abstract

Two manganese superoxide dismutase enzymes isolated from the eukaryote C. elegans have been characterized and their structures determined. The closely related structures reveal a striking similarity to manganese superoxide dismutase found in humans.

Partial Text

The superoxide dismutases (SODs; EC 1.15.11) are ubiquitous metalloproteins whose purpose is to detoxify the highly reactive superoxide anion (O2−) by its dismutation into oxygen and hydrogen peroxide (McCord & Fridovich, 1969 ▶). The superoxide anions would otherwise react with cell constituents, causing oxidative damage to macromolecules including lipids, DNA and proteins (Halliwell & Gutteridge, 1985 ▶). Since hydrogen peroxide, a product of the dismutation, is itself toxic to cells, the presence of SOD is intimately linked with that of catalase and peroxidase. Many organisms contain more than one type of SOD, distinguishable by their metal cofactor and subcellular location. Most eukaryotes, such as Caenorhabditis elegans, express both Cu/ZnSOD and MnSOD (Fridovich, 1975 ▶). Homologous FeSOD and MnSOD are expressed in prokaryotes (Bannister et al., 1987 ▶). C. elegans is a free-living nematode that lives in temperate soil environments. It serves as an interesting model to investigate SOD since it encodes five sod genes producing two cytosolic Cu/ZnSODs (SOD-1 and SOD-5), one extracellular Cu/ZnSOD (SOD-4) and two MnSODs (SOD-2 and SOD-3; designated MnSOD-2 and MnSOD-3, respectively) (C. elegans Genome Consortium, 1998 ▶). MnSOD-2 and MnSOD-3 are homologous proteins and are both synthesized as precursors with N-terminal mitochondrial targeting signals (Hunter et al., 1997 ▶). Extensive studies have revealed differential expression patterns, with MnSOD-2 emerging as the major constitutive MnSOD. MnSOD-3 is associated with diapause and its expression is induced in the long-lived dauer stage (Honda & Honda, 1999 ▶; Jones et al., 2001 ▶). Dissection of the insulin/IGF signalling pathway has identified sod-3 as a significant target for the DAF-16/FOXO transcription factor. Inhibition of the insulin/IGF signalling pathway results in elevated expression of sod-3 and an extension in lifespan (Murphy et al., 2003 ▶; Lee et al., 2003 ▶; Dong et al., 2007 ▶). Recently, it has been suggested that these MnSODs function in the insulin/IGF signalling pathway as physiological redox modulators rather than antioxidants (Honda et al., 2008 ▶). In a complementary approach to the study of differences in expression and cellular roles of the SODs of C. elegans, we are investigating the differences between the proteins themselves. Here we present the structures of the two MnSODs which are strikingly similar to that from human (Hearn et al., 2003 ▶).

Although MnSOD-2 and MnSOD-3 were overexpressed in E. coli without an N-terminal histidine tag, they each purified reasonably well using MCAC. A single-column procedure resulted in 66% purification for MnSOD-2 and 79% purification for MnSOD-3 as estimated by SDS–PAGE densitometry. A second MCAC purification yielded MnSOD-2 to 70% purity, while further ion exchange of MnSOD-3 using CM-52 gave a sample that was 98% pure. In a final step, gel-filtration chromatography resulted in 95% pure MnSOD-2 and 99% pure MnSOD-3 (results not shown). The subunit molecular weights of the purified proteins were measured by mass spectrometry and revealed that each of the MnSOD proteins had retained its N-­terminal methionine residue. Gel-filtration chromatography indicated that the pure proteins were tetrameric, a result that was confirmed by analytical ultracentrifugation (results not shown). Specific activity was estimated for MnSOD-2 as 3622 ± 80 U mg−1 per manganese and for MnSOD-3 as 3261 ± 26 U mg−1 per manganese; the proteins were 60 and 100% metallated with mangan­ese, respectively. These activities are comparable with that of the MnSOD from E. coli under the same conditions.

 

Source:

http://doi.org/10.1107/S1744309108037056

 

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