CRYSTALLIZATION AND PRELIMINARY X-RAY ANALYSIS OF A NITRATE REDUCTASE FROM DESULFOVIBRIO DESULFURICANS ATCC 27774. J. M. Dias1, C.Carneiro2, M. J. Almendra2, I. Moura2, J. Moura2, J. LeGall1,3, M. J. Romco1,4. 1ITQB, Apt 127, 2780 Oeiras, Portugal; 2FCT-UNL, 2785 Monte Caparica, Portugal; 3University of Georgia, Athens, GA 30602, USA; 4IST, Dept. Qummica, 1096 Lisboa Codex, Portugal
A nitrate reductase from Desulfovibrio (D.) desulfuricans ATCC 27774 (a sulfate reducing bacteria, that can adapt to nitrate respiration) has been isolated, allowing the elucidation of the enzymatic system required to convert nitrate (through nitrite) to ammonia. D. desulfuricans ATCC 27774 nitrate reductase is a monomeric periplasmic (soluble) enzyme with 74 kDa, containing one [4Fe-4S] centre and one molybdenum atom per molecule [1].
Crystals of this nitrate reductase were obtained by vapour diffusion with hanging drop technique using 6% (m/v) PEG 10 K as precipitant at pH 6.5 with 0.1M MES. The crystals grow at 20 :C or 4 :C to an approximate size of 0.3 x 0.3 x 0.3 mm.
The crystals diffract beyond 3.15 E resolution. Native data sets were collected using a MAR-Research Imaging Plate area detector and graphite monochromated Cu-Ka radiation from an Enraf Nonius rotating anode generator operated at 45kV and 99 mA. Data were processed with Denzo and the space group is P31,221 with cell parameters a=b= 106.9 E, c= 138.1 E.
The data set comprised 13201 measurements of 9695 independent reflections in the resolution range 30.0 < d < 3.15 E, with an overall completeness of 56.9 %, Rmerge(I)= 11.8 %.
1 - Bursakov, S., Liu, M., Payne, W., LeGall, J., Moura, I., Moura, J., Anaerobe, 1, 55-60, (1995).