Structural studies of the Trypanosoma cruzi Old Yellow Enzyme: insights into enzyme dynamics and specificity.

The flavoprotein old yellow enzyme of Trypanosoma cruzi (TcOYE) is an oxidoreductase that uses NAD(P)H as cofactor. This enzyme is clinically relevant due to its role in the action mechanism of some trypanocidal drugs used in the treatment of Chagas' disease by producing reactive oxygen species. In this work, the recombinant enzyme TcOYE was produced and collectively, X-ray crystallography, small angle X-ray scattering, analytical ultracentrifugation and molecular dynamics provided a detailed description of its structure, specificity and hydrodynamic behavior. The crystallographic structure at 1.27Å showed a classical (α/ß)8 fold with the FMN prosthetic group buried at the positively-charged active-site cleft. In solution, TcOYE behaved as a globular monomer, but it exhibited a molecular envelope larger than that observed in the crystal structure, suggesting intrinsic protein flexibility. Moreover, the binding mode of ß-lapachone, a trypanocidal agent, and other naphthoquinones was investigated by molecular docking and dynamics suggesting that their binding to TcOYE are stabilized mainly by interactions with the isoalloxazine ring from FMN and residues from the active-site pocket.

The stability and aggregation properties of the GTPase domain from human SEPT4.

The septins are a family of conserved proteins involved in cytokinesis and cortical organization. An increasing amount of data implicates different septins in diverse pathological conditions including neurodegenerative disorders, neoplasia and infections. Human SEPT4 is a member of this family and its tissue-specific ectopic expression profile in colorectal and urologic cancer makes it a useful diagnostic biomarker. Thermal unfolding of the GTPase domain of SEPT4 (SEPT4-G) revealed an unfolding intermediate which rapidly aggregates into amyloid-like fibers under physiological conditions. In this study, we examined the effects of protein concentration, pH and metals ions on the aggregation process of recombinant SEPT4-G using a series of biophysical techniques, which were also employed to study chemical unfolding and stability. Divalent metal ions caused significant acceleration to the rate of SEPT4-G aggregation. Urea induced unfolding was shown to proceed via the formation of a partially unfolded intermediate state which unfolds further at higher urea concentrations. The intermediate is a compact dimer which is unable to bind GTP. At 1 M urea concentration, the intermediate state was plagued by irreversible aggregation at temperatures above 30 degrees C. However, higher urea concentration resulted in a marked decay of the aggregation, indicating that the partially folded structures may be necessary for the formation of these aggregates. The results presented here are consistent with the recently determined crystal structure of human septins and shed light on the aggregation properties of SEPT4 pertinent to its involvement in neurodegenerative disease.

Crystallization and preliminary X-ray diffraction analysis of a glutathione S-transferase from Xylella fastidiosa.

Glutathione S-transferases (GSTs) form a group of multifunctional isoenzymes that catalyze the glutathione-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GST from Xylella fastidiosa (xfGST) was overexpressed in Escherichia coli and purified by conventional affinity chromatography. In this study, the crystallization and preliminary X-ray analysis of xfGST is described. The purified protein was crystallized by the vapour-diffusion method, producing crystals that belonged to the triclinic space group P1. The unit-cell parameters were a = 47.73, b = 87.73, c = 90.74 A, alpha = 63.45, beta = 80.66, gamma = 94.55 degrees. xfGST crystals diffracted to 2.23 A resolution on a rotating-anode X-ray source.

Crystallographic and pre-steady-state kinetics studies on binding of NADH to wild-type and isoniazid-resistant enoyl-ACP(CoA) reductase enzymes from Mycobacterium tuberculosis.

An understanding of isoniazid (INH) drug resistance mechanism in Mycobacterium tuberculosis should provide significant insight for the development of newer anti-tubercular agents able to control INH-resistant tuberculosis (TB). The inhA-encoded 2-trans enoyl-acyl carrier protein reductase enzyme (InhA) has been shown through biochemical and genetic studies to be the primary target for INH. In agreement with these results, mutations in the inhA structural gene have been found in INH-resistant clinical isolates of M.tuberculosis, the causative agent of TB. In addition, the InhA mutants were shown to have higher dissociation constant values for NADH and lower values for the apparent first-order rate constant for INH inactivation as compared to wild-type InhA. Here, in trying to identify structural changes between wild-type and INH-resistant InhA enzymes, we have solved the crystal structures of wild-type and of S94A, I47T and I21V InhA proteins in complex with NADH to resolutions of, respectively, 2.3A, 2.2A, 2.0 A, and 1.9A. The more prominent structural differences are located in, and appear to indirectly affect, the dinucleotide binding loop structure. Moreover, studies on pre-steady-state kinetics of NADH binding have been carried out. The results showed that the limiting rate constant values for NADH dissociation from the InhA-NADH binary complexes (k(off)) were eleven, five, and tenfold higher for, respectively, I21V, I47T, and S94A INH-resistant mutants of InhA as compared to INH-sensitive wild-type InhA. Accordingly, these results are proposed to be able to account for the reduction in affinity for NADH for the INH-resistant InhA enzymes.