Crystal structures of Leptospira interrogans FAD-containing ferredoxin-NADP+ reductase and its complex with NADP+

Abstract

Background: Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron transfer between NADP(H) and the proteins ferredoxin or flavodoxin. A number of structural features distinguish plant and bacterial FNRs, one of which is the mode of the cofactor FAD binding. Leptospira interrogans is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. Leptospira interrogans FNR (LepFNR) displays low sequence identity with plant (34% with Zea mays) and bacterial (31% with Escherichia coli) FNRs. However, LepFNR contains all consensus sequences that define the plastidic class FNRs.

Results: The crystal structures of the FAD-containing LepFNR and the complex of the enzyme with NADP+, were solved and compared to known FNRs. The comparison reveals significant structural similarities of the enzyme with the plastidic type FNRs and differences with the bacterial enzymes. Our small angle X-ray scattering experiments show that LepFNR is a monomeric enzyme. Moreover, our biochemical data demonstrate that the LepFNR has an enzymatic activity similar to those reported for the plastidic enzymes and that is significantly different from bacterial flavoenzymes, which display lower turnover rates.

Conclusion: LepFNR is the first plastidic type FNR found in bacteria and, despite of its low sequence similarity with plastidic FNRs still displays high catalytic turnover rates. The typical structural and biochemical characteristics of plant FNRs unveiled for LepFNR support a notion of a putative lateral gene transfer which presumably offers Leptospira interrogans evolutionary advantages. The wealth of structural information about LepFNR provides a molecular basis for advanced drugs developments against leptospirosis.

Figure 1

FNRs sequence alignment. Sequence alignment of plant and bacterial FNR. Identity is highlighted by color intensity. Alignment was performed in MUSCLE[36] and figure prepared in ESPript[37]. The arrows in the top denote sheet regions in the structure and the coils represent helices in LepFNR structure.

Figure 2

FNRs Crystallographic models. (a) Crystal structure of LepFNR colored by crystallographic B-factors indicates the region of flexible loops. 2F_obs-F_calc electron density map contoured at 1.0 sigma is shown for FAD molecule. In the insert, the plot of average B-factor per residue in LepFNR. (b) Detailed view of the metal binding site between two chains of LepFNR. The metal was modeled as a zinc atom. (c) Crystal structure of Zea mays FNR (PDB entry 1JB9[23]), showing FAD molecule in the extended form. (d) Crystal structure of Azobacter vinelandii (PDB entry 1A8P[7]), showing FAD molecule in bended form. The figures were prepared using PYMOL[38].

Figure 3

LepFNR SAXS data. (a) Experimental solution scattering curve of LepFNR and theoretical scattering intensities. Desmeared experimental curve is shown as dots with error bars; theoretical scattering intensity from the DAM is given in continuous line; scattering intensity from the LepFNR monomer is shown as a broken line. An inset displays the correspondent Guinier plot. (b) Comparison of distance distribution functions for LepFNR. The p(r) curves obtained were calculated using the GNOM program, the experimentally derived distribution for LepFNR is shown as dots with error bars. Distributions were calculates for the DAM (continuous line) and a single monomer of LepFNR crystal structure (broken line). (c) Stereoviews showing the superposition of the high-resolution crystallographic monomer of LepFNR with the envelope obtained from the data SAXS (spheres). Middle and bottom images are rotated by 90° around the y and x axes, respectively, compared to the top image.

Figure 4

FAD binding to FNR. (a) Schematic representation of FAD binding to FNR, prepared using LIGPLOT[39]. (b) Stereo view of FAD binding site. Key residues in FAD binding are shown with sticks.

Figure 5

Comparison of LepFNR crystallographic models bound to FAD and NADP⁺. (a) Superposition of LepFNR (green) and LepFNR·NADP⁺ (blue) crystal structures. Electronic densities are contoured at 1.0 sigma for FAD andNADP⁺. (b) Schematic representation of NADP⁺ binding to LepFNR.