Recombinant production, crystallization and crystal structure determination of dihydroorotate dehydrogenase from Leishmania (Viannia) braziliensis.

The enzyme dihydroorotate dehydrogenase (DHODH) is a flavoenzyme that catalyses the oxidation of dihydroorotate to orotate in the de novo pyrimidine-biosynthesis pathway. In this study, a reproducible protocol for the heterologous expression of active dihydroorotate dehydrogenase from Leishmania (Viannia) braziliensis (LbDHODH) was developed and its crystal structure was determined at 2.12 Å resolution. L. (V.) braziliensis is the species responsible for the mucosal form of leishmaniasis, a neglected disease for which no cure or effective therapy is available. Analyses of sequence, structural and kinetic features classify LbDHODH as a member of the class 1A DHODHs and reveal a very high degree of structural conservation with the previously reported structures of orthologous trypanosomatid enzymes. The relevance of nucleotide-biosynthetic pathways for cell metabolism together with structural and functional differences from the respective host enzyme suggests that inhibition of LbDHODH could be exploited for antileishmanicidal drug development. The present work provides the framework for further integrated in vitro, in silico and in vivo studies as a new tool to evaluate DHODH as a drug target against trypanosomatid-related diseases.

In silico assessment of S100A12 monomer and dimer structural dynamics: implications for the understanding of its metal-induced conformational changes.

Changes in the concentration of different ions modulate several cellular processes, such as Ca(2+) and Zn(2+) in inflammation. Upon activation of immune system effector cells, the intracellular Ca(2+) concentration rises propagating the activation signal, leading to degranulation and generation of reactive oxygen species, which increases the Zn(2+) intracellular concentration as a consequence of the cellular antioxidant machinery. In this context, S100A12 is of special interest because it is a pro-inflammatory protein expressed in neutrophils whose structure and function are modulated by both Ca(2+) and Zn(2+). The current hypothesis about its mechanism of action was built based on biochemical and crystallographic data. However, there are missing connections between molecular structure and the way in which many events are concatenated at the triggering and along the inflammatory process. In this work we use molecular dynamics simulations to describe how variations in Zn(2+) and Ca(2+) concentrations modulate the structural dynamics of the calcium-free S100A12 dimer and monomer, which was not considered a part of the mechanism of action before. Our results suggest that (i) Zn(2+) have a determinant role in the dimerization step, as well as in the unbinding of the Na(+) complexed to the N-terminal EF-hand; (ii) the N-terminal EF-hand domain is the first to bind Ca(2+), and not the C-terminal, as usually accepted; and that (iii) Ca(2+) modulates the structural dynamics of H-III.