Experimental and computational characterization of the ferric uptake regulator from Aliivibrio salmonicida (Vibrio salmonicida).

The Ferric uptake regulator (Fur) is a global transcription factor that affects expression of bacterial genes in an iron-dependent fashion. Although the Fur protein and its iron-responsive regulon are well studied, there are still important questions that remain to be answered. For example, the consensus Fur binding site also known as the "Fur box" is under debate, and it is still unclear which Fur residues directly interact with the DNA. Our long-term goal is to dissect the biological roles of Fur in the development of the disease cold-water vibriosis, which is caused by the psychrophilic bacteria Aliivibrio salmonicida (also known as Vibrio salmonicida). Here, we have used experimental and computational methods to characterise the Fur protein from A. salmonicida (AS-Fur). Electrophoretic mobility shift assays show that AS-Fur binds to the recently proposed vibrio Fur box consensus in addition to nine promoter regions that contain Fur boxes. Binding appears to be dependent on the number of Fur boxes, and the predicted "strength" of Fur boxes. Finally, structure modeling and molecular dynamics simulations provide new insights into potential AS-Fur-DNA interactions.

The first structure of a cold-active catalase from Vibrio salmonicida at 1.96 A reveals structural aspects of cold adaptation.

The cold-adapted catalase from the fish-pathogenic bacterium Vibrio salmonicida (VSC) has recently been characterized and shown to be two times more catalytically efficient compared with catalase from the mesophilic human pathogen Proteus mirabilis [PMC; Lorentzen et al. (2006), Extremophiles, 10, 427-440]. VSC is also less temperature-stable, with a half-life of 5 min at 333 K compared with 50 min for PMC. This was the background for solving the crystal structure of the cold-adapted VSC to 1.96 A and performing an extensive structural comparison of VSC and PMC. The comparison revealed that the entrance (the major channel) leading to the catalytically essential haem group, is locally more flexible and slightly wider in VSC. This might explain the enhanced catalytic efficiency of the nearly diffusion-controlled degradation of hydrogen peroxide into water and molecular oxygen in VSC. The reduced thermal stability of the cold-adapted VSC may be explained by a reduced number of ion-pair networks. The four C-terminal alpha-helices are displaced in the structures, probably owing to missing ionic interactions in VSC compared with PMC, and this is postulated as an initiation site for unfolding the cold-adapted enzyme. VSC is the first crystal structure reported of a cold-adapted monofunctional haem-containing catalase.

Crystallization and preliminary X-ray diffraction analysis of a cold-adapted catalase from Vibrio salmonicida.

Catalase (EC 1.11.1.6) catalyses the breakdown of hydrogen peroxide to water and molecular oxygen. Recombinant Vibrio salmonicida catalase (VSC) possesses typical cold-adapted features, with higher catalytic efficiency, lower thermal stability and a lower temperature optimum than its mesophilic counterpart from Proteus mirabilis. Crystals of VSC were produced by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 98.15, b = 217.76, c = 99.28 A, beta = 110.48 degrees. Data were collected to 1.96 A and a molecular-replacement solution was found with eight molecules in the asymmetric unit.

Optimisation of the surface electrostatics as a strategy for cold adaptation of uracil-DNA N-glycosylase (UNG) from Atlantic cod (Gadus morhua).

Cold-adapted enzymes are characterised by an increased catalytic efficiency and reduced temperature stability compared to their mesophilic counterparts. Lately, it has been suggested that an optimisation of the electrostatic surface potential is a strategy for cold adaptation for some enzymes. A visualisation of the electrostatic surface potential of cold-adapted uracil-DNA N-glycosylase (cUNG) from Atlantic cod indicates a more positively charged surface near the active site compared to human UNG (hUNG). In order to investigate the importance of the altered surface potential for the cold-adapted features of cod UNG, six mutants have been characterised and compared to cUNG and hUNG. The cUNG quadruple mutant (V171E, K185V, H250Q and H275Y) and four corresponding single mutants all comprise substitutions of residues present in the human enzyme. A human UNG mutant, E171V, comprises the equivalent residue found in cod UNG. In addition, crystal structures of the single mutants V171E and E171V have been determined. Results from the study show that a more negative electrostatic surface potential reduces the activity and increases the stability of cod UNG, and suggest an optimisation of the surface potential as a strategy for cold-adaptation of this enzyme. Val171 in cod UNG is especially important in this respect.