Structural and mutational analyses of the Leptospira interrogans virulence-related heme oxygenase provide insights into its catalytic mechanism

© 2017 Soldano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Heme oxygenase from Leptospira interrogans is an important virulence factor. During catalysis, redox equivalents are provided to this enzyme by the plastidic-type ferredoxin-NADP+ reductase also found in L. interrogans. This process may have evolved to aid this bacterial pathogen to obtain heme-iron from their host and enable successful colonization. Herein we report the crystal structure of the heme oxygenase-heme complex at 1.73 Å resolution. The structure reveals several distinctive features related to its function. A hydrogen bonded network of structural water molecules that extends from the catalytic site to the protein surface was cleared observed. A depression on the surface appears to be the H+ network entrance from the aqueous environment to the catalytic site for O2 activation, a key step in the heme oxygenase reaction. We have performed a mutational analysis of the F157, located at the above-mentioned depression. The mutant enzymes were unable to carry out the complete degradation of heme to biliverdin since the reaction was arrested at the verdoheme stage. We also observed that the stability of the oxyferrous complex, the efficiency of heme hydroxylation and the subsequent conversion to verdoheme was adversely affected. These findings underscore a long-range communication between the outer fringes of the hydrogen-bonded network of structural waters and the heme active site during catalysis. Finally, by analyzing the crystal structures of ferredoxin-NADP+ reductase and heme oxygenase, we propose a model for the productive association of these proteins

A Highly Stable Plastidic-Type Ferredoxin-NADP(H) Reductase in the Pathogenic Bacterium Leptospira interrogans

Leptospira interrogans is a bacterium that is capable of infecting animals and humans, and its infection causes leptospirosis with a range of symptoms from flu-like to severe illness and death. Despite being a bacteria, Leptospira interrogans contains a plastidic class ferredoxin-NADP(H) reductase (FNR) with high catalytic efficiency, at difference from the bacterial class FNRs. These flavoenzymes catalyze the electron transfer between NADP(H) and ferredoxins or flavodoxins. The inclusion of a plastidic FNR in Leptospira metabolism and in its parasitic life cycle is not currently understood. Bioinformatic analyses of the available genomic and proteins sequences showed that the presence of this enzyme in nonphotosynthetic bacteria is restricted to the Leptospira genus and that a [4Fe-4S] ferredoxin (LB107) encoded by the Leptospira genome may be the natural substrate of the enzyme. Leptospira FNR (LepFNR) displayed high diaphorase activity using artificial acceptors and functioned as a ferric reductase. LepFNR displayed cytochrome c reductase activity with the Leptospira LB107 ferredoxin with an optimum at pH 6.5. Structural stability analysis demonstrates that LepFNR is one of the most stable FNRs analyzed to date. The persistence of a native folded LepFNR structure was detected in up to 6 M urea, a condition in which the enzyme retains 38% activity. In silico analysis indicates that the high LepFNR stability might be due to robust interactions between the FAD and the NADP+ domains of the protein. The limited bacterial distribution of plastidic class FNRs and the biochemical and structural properties of LepFNR emphasize the uniqueness of this enzyme in the Leptospira metabolism. Our studies show that in L. interrogans a plastidic-type FNR exchanges electrons with a bacterial-type ferredoxin, process which has not been previously observed in nature

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

<p>Abstract</p> <p>Background</p> <p>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. <it>Leptospira interrogans </it>is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. <it>Leptospira interrogans </it>FNR (LepFNR) displays low sequence identity with plant (34% with <it>Zea mays</it>) and bacterial (31% with <it>Escherichia coli</it>) FNRs. However, LepFNR contains all consensus sequences that define the plastidic class FNRs.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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 <it>Leptospira interrogans </it>evolutionary advantages. The wealth of structural information about LepFNR provides a molecular basis for advanced drugs developments against leptospirosis.</p

Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans

Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP+ reductase

Reduction of the ferric LepHO-heme complex in anaerobic conditions and spontaneous reoxidation.

<p>Time dependent formation (A) and autoxidation (B) of the ferrous heme complex of wild type LepHO (●) and F157I mutant (○) as monitored by variations in absorbance at 426 and 403 nm, respectively. Data extracted from the spectra shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182535#pone.0182535.g005" target="_blank">Fig 5</a>.</p

LepHO heme binding pocket and hydrogen bond network.

<p>A) Distinctive hydrophobic residues facing the α-meso carbon atom of heme in LepHO. B) LepHO residues, water molecules (blue spheres) and coordination bonds (broken lines) involved in the hydrogen bond distal site network. C) Detailed view of a LepHO structure in surface representation, where it can be seen that the hydrogen bonded network of structural waters reaches the surface of the protein and suggests a possible proton entry site.</p

Absorption spectral changes of the LepHO-heme complex during the NADPH/LepFNR-supported heme degradation.

<p>Time dependent absorption spectra of wild type LepHO (A), F157I (B) and F157A (C), before (---) and after (―) the addition of LepFNR and NADPH: Experimental conditions are as indicated in Materials and methods. The inset shows an enlargement of the spectral region between 500 and 800 nm. The time-dependent decay of the intensity at 403 nm (D) and the increase at 680 nm (E) were obtained from the spectra shown in panels (A) to (C). Wild type LepHO (●); F157I (▼) and F157A (○).</p