Identification of succinic semialdehyde reductases from Geobacter: expression, purification, crystallization, preliminary functional, and crystallographic analysis.
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.
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, 48824-1319, USA.
Yanfeng
Y
Zhang
Xiaoli
X
Gao
Yi
Y
Zheng
R Michael
RM
Garavito
D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods*; D045302:Geobacter / Q000201:enzymology* / Q000378:metabolism; D006884:Hydroxybutyrate Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D006884:Hydroxybutyrate Dehydrogenase; C019416:4-hydroxybutyrate dehydrogenase