The ß-hairpin from the Thermus thermophilus HB27 laccase works as a pH-dependent switch to regulate laccase activity.

The multi-copper oxidase from the hyper-thermophilic bacteria Thermus thermophilus (Tth-MCO), has been previously characterized and described as an example of a laccase with low catalytic properties, especially when it is compared with the activity of fungal laccases, but it is active at high temperatures. Structurally, Tth-MCO has a unique feature: a ß-hairpin near the T1Cu site, which is not present in any other laccases deposited at the PDB. This ß-hairpin has an expected crystallographic behavior in solvent-exposed areas of a crystallized protein: lack of electron density, high B-values and several crystalline contacts with neighboring crystallographic copies; however, its dynamical behavior in solution and its biological implications have not been described. Here, we describe four new Tth-MCO crystallographic structures, and the ß-hairpin behavior has been analyzed by molecular dynamics simulations, considering the effect of pH and temperature. The ß-hairpin new crystallographic conformations described here, together with their dynamics, were used to understand the pH-restrained laccase activity of Tth-MCO against substrates as syringaldazine. Remarkably, there are insertions in laccases from Thermus and Meiothermus genus, sharing the same position and a methionine-rich composition of the Tth-MCO ß-hairpin. This unique high methionine content of the Tth-MCO ß-hairpin is responsible to coordinate, Ag+1 and Hg+1 in oxidative conditions, but Cu+1 and Cu+2 are not coordinated in crystallographic experiments, regardless of the redox conditions; however, Ag+1 addition does not affect Tth-MCO laccase activity against syringaldazine. Here, we propose that the pH-dependent ß-hairpin dynamical behavior could explain, at least in part, the inefficient laccase activity displayed by Tth-MCO in acidic pH values.

Cloning, recombinant production and crystallographic structure of Proliferating Cell Nuclear Antigen from radioresistant archaeon Thermococcus gammatolerans.

Thermococcus gammatolerans is a strictly anaerobic; hyperthermophilicarchaeon belongs to the order Thermococcales in the phylum Euryarchaeota. It was extracted from a hydrothermal vent from the Guaymas Basin (Gulf of California, Mexico). Different studies show that T. gammatolerans is one of the most radioresistant organisms known amongst the archaea. This makes it a unique model to study adaptations to the environment and to study DNA repair mechanisms in an organism able to tolerate harsh conditions. A key protein in these mechanisms is the Proliferation Cell Nuclear Antigen (PCNA). Its function is focused on their ability to slide along the DNA duplex and coordinating the activities of proteins mainly related to DNA edition and processing. Analysis of archaeal proteins have proven to be enormously fruitful because much of the information obtained from them can be extrapolated to eukaryotic systems, and PCNA is no exception. Here we report the cloning, recombinant expression and crystallographic structure of PCNA from T. gammatolerans (TgPCNA).

Structural flexibility, an essential component of the allosteric activation in Escherichia coli glucosamine-6-phosphate deaminase.

A new crystallographic structure of the free active-site R conformer of the allosteric enzyme glucosamine-6-phosphate deaminase from Escherichia coli, coupled with previously reported structures of the T and R conformers, generates a detailed description of the heterotropic allosteric transition in which structural flexibility plays a central role. The T conformer's external zone [Horjales et al. (1999), Structure, 7, 527-536] presents higher B values than in the R conformers. The ligand-free enzyme (T conformer) undergoes an allosteric transition to the free active-site R conformer upon binding of the allosteric activator. This structure shows three alternate conformations of the mobile section of the active-site lid (residues 163-182), in comparison to the high B values for the unique conformation of the T conformer. One of these alternate R conformations corresponds to the active-site lid found when the substrate is bound. The disorder associated with the three alternate conformations can be related to the biological regulation of the K(m) of the enzyme for the reaction, which is metabolically required to maintain adequate concentrations of the activator, which holds the enzyme in its R state. Seven alternate conformations for the active-site lid and three for the C-terminus were refined for the T structure using isotropic B factors. Some of these conformers approach that of the R conformer in geometry. Furthermore, the direction of the atomic vibrations obtained with anisotropic B refinement supports the hypothesis of an oscillating rather than a tense T state. The concerted character of the allosteric transition is also analysed in view of the apparent dynamics of the conformers.