Download Acidaminococcus fermentans

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NAD -dependent (R)-2-Hydroxyglutarate Dehydrogenase from
Acidaminococcus fermentans
B.M. Martins1, S. Macedo-Ribeiro1*, J. Bresser2, R. Huber1, W. Buckel2 and A. Messerschmidt1
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Max-Planck-Institut fuer Biochemie, Am Klopferspitz 18-a, 82152 Martinsried by Muenchen, Germany
Fachbereich Biologie-Mikrobiologie, Philipps-Universitaet Marburg, Karl-von-Frisch. Str., 35032 Marburg, Germany
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Present address: Instituto de Biologia Molecular de Barcelona, Carrer Jordi Girona 18-26, 08034 Barcelona, Spain
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The strictly anaerobe Acidaminococcus fermentans is a non-pathogenic clostridial bacterium commonly
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found in mammals‘ digestive track. The NAD -dependent (R)-2-Hydroxyglutarate Dehydrogenase (HGDH)
catalyses the second step in the glutamate fermentation via hydroxyglutarate, converting α-ketoglutarate to
(R)-2-hydroxyglutarate. HGDH belongs to the D-specific 2-hydroxyacid dehydrogenases family. Their
members have considerable biotechnological potential, both for the chiral synthesis of novel nonprotegenic
amino acids for use in the pharmaceutical industry and for use as diagnostic reagents to monitor serum
levels of amino acid which accumulate in a range of metabolic diseases. Additionally, the analysis of the
three-dimensional structure may help to clarify the enzymatic basis for a progressive neurodegenerative
disease called (R)-2-Hydroxyglutarate Aciduria.
HGDH was crystallised by the sitting drop vapour diffusion method at 4°C. Additionally, co-crystallisation
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trials have been performed with cofactor (NAD ), substrate (2-oxoglutarate) or competitive inhibitor
(oxalylglycine, a substrate analogue). Rod-shaped crystals appear after 10 to 15 days with approximate
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dimensions of 700x400x400 µm . They belong to the hexagonal space group P6122 with cell constants a = b
= 68.79 Å, c = 313.13 Å, and α = β = 90°, γ = 120°. There is one molecule per asymmetric unit and the unit
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cell contains 52% solvent content (Vm = 2.58 Å /Dalton). Whereas in the house x-ray facility (MAR IP
detector) diffraction data could only be collected to 2.8 Å due to the c axis dimension, it was possible to
extend the data resolution up to 1.98 Å using a MAR IP detector at the DESY synchrotron beamline BW6.
The crystals were left for 5 to 10 minutes in a harvesting buffer containing 10 to 15 % glycerol as
cryoprotectant prior to flash cooling in the nitrogen gas liquid at 4°C. A diffraction pattern from a crystal
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co-crystallised in the presence of NAD is shown in figure 1.
Figure 1: The resolution at the edge of the image is 1.98 •.
References
[1] J. Bresser, Diplomarbeit, Fachbereich Biologie-Mikrobiologie, Philipps-Universitaet Marburg,
Germany (1997).
[2] W. Buckel and H.A. Barker, J. Bacteriol. 117, 1248 (1974).