Identification of succinic semialdehyde reductases from Geobacter: expression, purification, crystallization, preliminary functional, and crystallographic analysis.

Yanfeng Zhang, Xiaoli Gao, Yi Zheng, R Michael Garavito

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, 48824-1319, USA.

Acta biochimica et biophysica Sinica 2011 Dec

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Succinic semialdehyde reductase (SSAR) is an important enzyme involved in γ-aminobutyrate (GABA) metabolism. By converting succinic semialdehyde (SSA) to γ-hydroxybutyrate (GHB), the SSAR facilitates an alternative pathway for GABA degradation. In this study, we identified SSARs from Geobacter sulfurreducens and Geobacter metallireducens (GsSSAR and GmSSAR, respectively). The enzymes were over-expressed in Escherichia coli and purified to near homogeneity. Both GsSSAR and GmSSAR showed the activity of reducing SSA using nicotinamide adenine dinucleotide phosphate as a co-factor. The oligomeric sizes of GsSSAR and GmSSAR, as determined by analytical size exclusion chromatography, suggest that the enzymes presumably exist as tetramers in solution. The recombinant GsSSAR and GmSSAR crystallized in the presence of NADP(+), and the resulting crystals diffracted to 1.89 Å (GsSSAR) and 2.25 Å (GmSSAR) resolution. The GsSSAR and GmSSAR crystals belong to the space groups P2(1)22(1) (a= 99.61 Å, b= 147.49 Å, c= 182.47 Å) and P1 (a= 75.97 Å, b= 79.14 Å, c= 95.47 Å, α = 82.15°, β = 88.80°, γ = 87.66°), respectively. Preliminary crystallographic data analysis suggests the presence of eight protein monomers in the asymmetric units for both GsSSAR and GmSSAR.

Citation

Yanfeng Zhang, Xiaoli Gao, Yi Zheng, R Michael Garavito. Identification of succinic semialdehyde reductases from Geobacter: expression, purification, crystallization, preliminary functional, and crystallographic analysis. Acta biochimica et biophysica Sinica. 2011 Dec;43(12):996-1002