ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
P29. 1.5 Å Crystal Structure of the Heterodimeric Nitrate Reductase from
Cupriavidus necator
C. Coelho, P. J.González, J. Trincão, A. L.Carvalho, S. Najmudin, I. Moura,
J. J. G. Moura and M. João Romão
REQUIMTE, Departamento de Química, CQFB, FCT-UNL, 2829-516 Caparica, Portugal,
e-mail: catarinacoelho@dq.fct.unl.pt
Nitrate Reductases belong to the DMSO reductase family of mononuclear Mo-containing enzymes. Periplasmic
nitrate reductase (Nap) catalyses the reduction of nitrate to nitrite. This enzyme is responsible for initiating
anaerobic ammonification and also participates in the cellular redox balancing, by which nitrate is used to
dissipate excess reducing power [1]. Until recently only three crystal structures of Nap were available: NapA
from Desulfovibrio desulfuricans (Dd NapA, to 1.9 Å), NapA from Escherichia coli (E.coli NapA, to 2.5 Ǻ) and
NapAB from Rhodobacter sphaeroides (Rs NapAB, to 3.2 Å) [2, 3, 4]. We report here the crystal structure of a
heterodimeric Nap from Cupriavidus necator (formerly Ralstonia eutropha). CnNapAB comprises a 91 kDa
catalytic subunit (NapA) and a 17 kDa subunit (NapB) involved in electron transfer. The larger subunit contains
a molybdenum active site with a bis-molybdopterin guanine dinucleotide cofactor as well as one [4Fe–4S]
cluster, while the small subunit is a di-haem c-type cytochrome. Crystals of the oxidized form of this enzyme
were obtained using PEG 3350 as precipitant. A single crystal grown at the High Throughput Crystallization
Laboratory of the EMBL in Grenoble diffracted to beyond 1.5 Å at the ESRF (ID14-1), the highest resolution
reported to date for a nitrate reductase. The unit-cell parameters are a = 142.2, b = 82.4, c = 96.8 Å and β=
100.7º, space group C2, and one heterodimer is present per asymmetric unit [5]. One clear solution was obtained
by Molecular Replacement using DdNap as a search model and the structure was refined to a final R factor/Rfree
of 0.17/0.20.
References:
[1] J.J.G. Moura, C.D. Brondino, J. Trincão and M J. Romão, J Biol Inorg Chem, 9, 791 (2004).
[2] P. Arnoux, M. Sabaty, J. Alric, B. Frangioni, B. Guigliarelli, J. Adriano and D. Pignol, Nature Structural
Biology, 10, 928 (2003).
[3] J.M. Dias, M.E. Than, A. Humm, R. Huber, G.P. Bourenkov, H.D. Bartunik, S. Bursakov, J. Calvete, J.
Caldeira, C. Carneiro, J.J.G. Moura, I. Moura and M.J. Romão, Structure, 7, 65 (1999).
[4] B J.N. Jepson, S. Mohan, T.A. Clarke., A.J. Gates, J.A. Cole, C.S. Butler, J.N. Butt, A.M. Hemmings, D.J.
Richardson, J. Biol. Chem., 282, 6425 (2007).
[5] C. Coelho, P.J. González, J. Trincão, A.L.Carvalho, S. Najmudin, I. Moura, J.J.G. Moura, T. Hettman, S.
Dieckman and M.J. Romão, Acta Cryst., F63, 516 (2007).
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