Structure of the Month: April 2009 [see all]
Structural basis of the catalytic mechanism of gluconate 5-dehydrogenase from Streptococcus suis
Qiangmin Zhang1,2, Hao Peng1, Feng Gao1,3, Yiwei Liu1, Hao Cheng1,2 John Thompson4 and George F. Gao1,2,5,6*
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Streptococcus suis serotype 2 (SS2) is a an emerging pathogen with zoonotic importance causing diseases in pigs and humans, that has generated world-wide public concern (Tang et al. 2006). Potent bactericidal inhibitors will be required for effective treatment and prevention of S. suis infection in humans because there is no vaccine available for human beings. Gluconate 5-dehydrogenase (Ga5DH; EC 1.1.1.69) is only found in bacterial species and not in humans, and therefore thought to be a potential target for therapy of human S. suis infection. Ga5DH, catalyzing a reversible oxidoreduction between D-gluconate and 5-keto-D-gluconate, is also an uncharacterized member of short dehydrogenase/reductase (SDR) family.
To this end, we cloned and expressed this protein in E. coli BL21. Subsequently we obtained the crystals of Ga5DH in native and complexed forms. The X-data of native and complexed Ga5DH were collected to 1.8 and 1.9 Å resolution, respectively. The S. suis Ga5DH is a homotetramer in solution, containing two distinct domains: the N-terminal domain, responsible for NADP+ binding, and the C-terminal domain that harbors the catalytically important triad of residues Ser-Tyr-Lys, as is the case in other members of SDR enzymes (Jornvall et al. 1995).
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Figure 1: The cartoon diagram of Ga5DH monomer. The N-terminal domain is colored in white and C-terminal domain in green. The cofactor NADP+ (white stick), calcium ion (magenta sphere) and substrate gluconate (yellow stick) with well-defined electron density (represented by cyan mesh) are located between these two domains. |
In the crystal structure of the Ga5DH complex, we found that a charged residue Arg104 forms a very strong hydrogen bond with the substrate. This residue possibly participates in the recognition and stabilization of the substrate, and the proposal is confirmed by site-directed mutagenesis in our sturdy. Accordingly, we proposed a catalytically active tetrad RYSK in S. suis Ga5DH as well as a plausible mechanism.
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Figure 2: The catalytically important residues in S. suis Ga5DH. The substrate is depicted as a yellow stick model. |
This work was performed in the Laboratory of Prof. George F. Gao in the CAS Key Laboratory of Pathogenic Microbiology and Immunology (CASPMI), Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing, P.R. China.
Data collection details
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Sample |
Native |
Complex |
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PDB ID |
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Space group |
I222 |
I222 |
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Unit cell |
a =71.1 Å, b = 75.6 Å, c = 97.8 Å |
a =71.2 Å, b = 75.3 Å, c = 98.7 Å |
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Radiation |
Cu Kα |
Cu Kα |
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Generator |
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Optic |
Blue |
Blue |
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Detector |
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Crystal-to-detector distance |
120 mm |
120 mm |
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Exposure time per frame |
3 min |
3 min |
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Oscillation width |
1° |
1° |
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Number of frames |
180 |
230 |
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Data Processing |
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Resolution range |
50.0 – 1.8 Å |
50.0 – 1.9 Å |