Zo-peroxidase: Crystal structure and sequence of a highly-glycosylated peroxidase resistant to high concentrations of H2O2 from Japanese radish

Understanding Peroxidase (PRXs) enzymatic diversity and functional significance from a three-dimensional point of view is a key point for structural and mechanistic studies. In this context, Zo-peroxidase (ZoPrx) a member of the class III peroxidases and secreted by plants, differs from all previously described PRXs because of its remarkable catalytic stability in the presence of hydrogen peroxide. In this work, we present the crystallographic structure of ZoPrx isolated from Japanese radish, at 2.05 Å resolution. The mature enzyme consists of a single monomer of 308 residues exhibiting the same fold as all previously described members of the plant PRXs superfamily. Furthermore, the enzyme contains a heme b group as the prosthetic group and two Ca2+ binding sites. Moreover, seven N-glycosylation sites were found in the structure, and 49 glycans bound to the two ZoPrx molecules found in the asymmetric unit are clearly visible in the electron density map. The comparison of ZoPrx coordinates with homologous enzymes revealed minor structural changes, in which the residue 177 appears to be responsible for enlarging the access to the heme cavity, the only structural finding which may be related to the H2O2 tolerance of ZoPrx and detected by X-ray crystallography. Because of its characteristics, ZoPrx has a broad range of potential applications from chemical synthesis to environmental biocatalysis, thus its aminoacidic sequence, partially completed using the electron density, and the three-dimensional structure itself, become a possible starting point to engineering heme-peroxidases to enhance oxidative stability. Keywords: Glycosylated protein structure, Hydrogen peroxide tolerance, Oxidase, Plant peroxidase, Redox enzym

A Putative New Role of Tv-PSP1 Recognizes IRE and ERE Hairpin Structures from <i>Trichomonas vaginalis</i>

To understand whether protein Tv-PSP1 from Trichomonas vaginalis recognizes mRNA parasite stem-loop structures, we conducted REMSA and intrinsic fluorescence assays. We found the recombinant Tv-PSP1 structure, determined with X-ray crystallography, showed unusual thermal stability of the quaternary structure, associated with a disulfide bridge CYS76-CYS104. To gain deeper insight into the Tv-PSP1 interaction with mRNA stem-loops (mRNAsl) and its relationship with thermal stability, we also used an integrated computational protocol that combined molecular dynamics simulations, docking assays, and binding energy calculations. Docking models allowed us to determine a putative contact surface interaction region between Tv-PSP1 and mRNAsl. We determined the contributions of these complexes to the binding free energy (ΔGb) in the electrostatic (ΔGelec) and nonelectrostatic (ΔGnon-elec) components using the Adaptive Poisson–Boltzmann Solver (APBS) program. We are the first, to the best of our knowledge, to show the interaction between Tv-PSP1 and the stem-loop structures of mRNA

Characterization of a catalase-peroxidase variant (L333V-KatG) identified in an INH-resistant Mycobacterium tuberculosis clinical isolate

Mycobacterium tuberculosis catalase-peroxidase (Mt-KatG) is a bifunctional heme-dependent enzyme that has been shown to activate isoniazid (INH), the widely used antibiotic against tuberculosis (TB). The L333V-KatG variant has been associated with INH resistance in clinical M. tuberculosis isolates from Mexico. To understand better the mechanisms of INH activation, its catalytic properties (catalase, peroxidase, and IN-NAD formation) and crystal structure were compared with those of the wild-type enzyme (WT-KatG). The rate of IN-NAD formation mediated by WT-KatG was 23% greater than L333V-KatG when INH concentration is varied. In contrast to WT-KatG, the crystal structure of the L333V-KatG variant has a perhydroxy modification of the indole nitrogen of W107 from MYW adduct. L333V-KatG shows most of the active site residues in a similar position to WT-KatG; only R418 is in the R-conformation instead of the double R and Y conformation present in WT-KatG. L333V-KatG shows a small displacement respect to WT-KatG in the helix from R385 to L404 towards the mutation site, an increase in length of the coordination bond between H270 and heme Fe, and a longer H-bond between proximal D381 and W321, compared to WT-KatG; these small displacements could explain the altered redox potential of the heme, and result in a less active and stable enzyme