Cloning, expression, crystallization and preliminary X-ray studies of the ferredoxin-NAD(P)+ reductase from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

Abstract

Ferredoxin-NADP(+) reductase (FNR) is a flavoenzyme that catalyses the reduction of NADP(+) in the final step of the photosynthetic electron-transport chain. FNR from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TeFNR) contains an additional 9 kDa domain at its N-terminus relative to chloroplastic FNRs and is more thermostable than those from mesophilic cyanobacteria. With the aim of understanding the structural basis of the thermostability of TeFNR and assigning a structural role to the small additional domain, the gene encoding TeFNR with and without an additional domain was engineered for heterologous expression and the recombinant proteins were purified and crystallized. Crystals of TeFNR without the additional domain belonged to space group P2(1), with unit-cell parameters a = 55.05, b = 71.66, c = 89.73 Å, α = 90, β = 98.21, γ = 90°.

Figure 1

A typical crystal of T. elongatus FNR without the CpcD-like domain.

Figure 2

A diffraction image from truncated TeFNR recorded at the Photon Factory.

Figure 3

SDS–PAGE analysis of full-length FNR. (a) Freshly purified FNR was applied in the middle lane. Dissolved crystals of TeFNR without any protein inhibitor were applied in the right lane. (b) Dissolved crystals prepared in the presence of 10 mM PMSF were applied to the right gel. The left lanes contain molecular-mass markers (labelled in kDa).

Figure 4

Molecular packing drawn as a stereo pair projected along the c axis. The asymmetric unit contains two molecules.