Fig. 1. The Cα polypeptide backbones and FAD conformations of plant-type FNRs. The computer graphics are based on X-ray diffraction data for the spinach leaf (A), Anabaena PCC7119 (B), E. coli (C) and A. vinelandii (D) flavoenzymes. The N-terminal FAD binding domain is shown in red, the C-terminal NADP(H) binding domain in blue, and the prosthetic group in yellow. The arrow indicates the sheet-loop-sheet motif characteristic of the plastidic FNR class.

Fig. 2. Phylogenetic relationships within the plastidic FNR class. The trees were constructed from 35 sequences with 348 aligned positions using Kimura protein distance metric [76] and the Neighbor-joining clustering method [77], both implemented in the phylogeny inference package PHYLIP Version 3.6 (alpha3) [78]. The multiple protein sequence alignments used to calculate the pairwise distance matrices were generated with the program ClustalX v1.83 [78]. Bootstrap values after 1000 resamplings are given in percentages and indicated at the corresponding branch. The distance between two sequences can be obtained by the additions of the lengths of the connecting horizontal branches, using the scale on top. Letters between brackets indicate reductases from leaf (L) or root (R).

Fig. 3. Phylogenetic relationships within the bacterial FNR class. The distance trees were constructed from 54 sequences with 283 aligned positions as indicated in the legend to Fig. 2. The “P” between brackets indicates reductases from proteobacteria.

Fig. 4. Conserved sequences in plant-type FNRs. The six peptide segments (1–6) that define this structural family are aligned, showing in bold the amino acids involved in FAD (boxes 1, 2 and 6) or NADP(H) binding (boxes 3–5). The residues contained in box “A” interact with the adenosine moiety of FAD and are specific of the plastidic FNR class, whereas those in box “B” allow discrimination of the bacterial reductases in subclass I (A. vinelandii) and II (E. coli). Numerals above the residues indicate their relative positions in the spinach FNR sequence. Detailed views of FAD conformations and relevant active site interactions in three FNR prototypes (Spinacia oleracea, A. vinelandii and E. coli) are shown on the right side.

Table 1. Sorting of plant-type FNRs by primary and tertiary structure, FAD conformation and catalytic ratesa