Crystal structures of dimethylarginine dimethylaminohydrolase-1 (DDAH-1) from Homo sapiens bound to the inhibitors N⁵-(1-iminopentyl)-L-ornithine and ebselen and functional studies of the translin●trax complex from Mus musculus

Nitric oxide (NO) is reactive, radical gas that is involved in a myriad of cellular signaling pathways including the regulation of blood flow and immunodefense. NO is produced from the oxidation of L-arginine to L-citrulline by nitric oxide synthase (NOS). The activity of NOS and by default, the production of NO, is regulated by the arginine derivatives N[omega],N[omega]-dimethyl-L-arginine (ADMA) and N[omega]-monomethyl-L-arginine (NMMA) which arise from the proteolytic degradation of post translationally methylated proteins. The cellular concentrations of ADMA and NMMA are regulated by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which catabolizes these compounds to L-citrulline and dimethylamine or methyl amine, respectively. Because over and under production of NO has been implicated in several pathophysiological states, compounds that control NO production by inhibiting NOS or DDAH may prove useful as treatments. In this study, the crystal structures of human DDAH-1 with the inhibitors N⁵-(1-iminopentyl)-L-ornithine (L-IPnO) and ebselen were solved to 2.9 and 2.0 Å resolution, respectively. L-IPnO was observed to inhibit DDAH-1 in essentially the same manner as another amidino-containing inhibitor: docking to the enzyme via hydrogen bond and ion pair interactions and forming a covalent adduct with the active site cysteine. Ebselen was also observed to covalently attach to the active site cysteine, however, the docking mechanism was absent of hydrogen bond and ion pair interactions. The work presented here contributes to the design of compounds that may effectively regulate the production of NO for therapeutic purposes. Translin is a highly conserved mammalian RNA and DNA binding protein known to be involved in DNA recombination and repair, RNA trafficking in neurons, and post-transcriptional regulation of gene expression in male germ cells. Although crystal structures of the mouse and human orthologs of translin have been solved, they do not provide details on the structure-function relationship of the protein. Studies have identified a partner protein for translin, translin associated factor x (trax), which is believed to have a crucial role in assisting translin with its cellular functions. It is believed that trax regulates translin’s affinity for certain RNA and DNA sequences. In this work the binding affinities of translin and the translin●trax complex were investigated. It was observed that translin preferentially binds to G-rich RNA sequences, most likely recognizing a secondary structure intrinsic to these sequences, whereas translin●trax preferentially binds G-rich DNA sequences. The results from these experiments provide insight into the cellular functions of translin and trax and their respective roles in mRNA trafficking.