Cloning, expression, crystallization and preliminary X-ray studies of the ferredoxin–NAD(P)⁺reductase from the thermophilic cyanobacteriumThermosynechococcus elongatusBP-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°.

1 Cyanobacterial design cell for the production of hydrogen from water

“…The residue exchange from basic lysine to acid aspartate at K75D in the FNR S of T. elongatus shows a 20 to 30% reduced activity towards Fd in comparison to WT-FNR s , resulting in an up to 80 % increase of the H 2 production rate (Figure 6c). This is an excellent basis for further reduction of the Fd-FNR interaction in the design cell of Synechocystis, especially if the generation of double mutants based on the available 3D structural analysis is considered [27]. In summary, these in vitro experiments show that a considerable re-routing of electrons at the acceptor side of PS1 is possible in favor of a future hydrogen production.…”

“…short version). According to previous studies (Anabaena PCC 7119 [29]; Zea mays [30], T. elongatus [27]), the amino acid residue 75 (acc. to the FNR S sequence) was selected as the target for site-directed mutagenesis.…”

“…Detailed structural knowledge of the Fd-binding sites of FNR and H 2 ase enables the production of directed mutants which -in an iterative process -allows a stepwise decrease in the affinity for FNR [27] while keeping or even increasing the affinity for H 2 ase. The success of these studies was first evaluated by in vitro studies using overexpressed Fd and FNR variants from the thermophilic cyanobacterium Thermosynechococcus elongatus (T. elongatus).…”

Metabolic engineering of cyanobacteria for the production of hydrogen from water

“…Besides the construction of Fdhydrogenase fusion proteins [16], one strategy to manipulate electron partitioning between both enzymes is to engineer the FNR-binding site for lower Fd affinity. Owing to detailed 3D structures being available for both Fd and various FNR mutants [17], a rational design is possible. Preliminary functional characterization of several mutants showed that K d values could be shifted by a factor of more than 10 as determined with isolated FNR from WT and directed mutants (K. Wiegand and K. Cormann, personal communication).…”

Light‐Driven NADPH Cofactor Recycling by Photosystem I for Biocatalytic Reactions

“…The ultimate electron source for this process in the natural system is the water‐splitting activity of photosystem II (PSII). Here, we developed the proof‐of‐concept for a light‐driven in vitro system for NADPH recycling by coupling of isolated PSI, [8] Fd, [9] FNR, [10] with three different oxidoreductases (Figure 1).…”