Crystal structure of thebaine 6-O-demethylase from the morphine biosynthesis pathway.

Thebaine 6-O-demethylase (T6ODM) from Papaver somniferum (opium poppy), which belongs to the non-heme 2-oxoglutarate/Fe(II)-dependent dioxygenases (ODD) family, is a key enzyme in the morphine biosynthesis pathway. Initially, T6ODM was characterized as an enzyme catalyzing O-demethylation of thebaine to neopinone and oripavine to morphinone. However, the substrate range of T6ODM was recently expanded to a number of various benzylisoquinoline alkaloids. Here, we present crystal structures of T6ODM in complexes with 2-oxoglutarate (T6ODM:2OG, PDB: 5O9W) and succinate (T6ODM:SIN, PDB: 5O7Y). Both metal and 2OG binding sites display similarity to other proteins from the ODD family, but T6ODM is characterized by an exceptionally large substrate binding cavity, whose volume can partially explain the promiscuity of this enzyme. Moreover, the size of the cavity allows for binding of multiple molecules at once, posing a question about the substrate-driven specificity of the enzyme.

On the Structure and Reaction Mechanism of Human Acireductone Dioxygenase.

Acireductone dioxygenase (ARD) is an intriguing enzyme from the methionine salvage pathway that is capable of catalysing two different oxidation reactions with the same substrate depending on the type of the metal ion in the active site. To date, the structural information regarding the ARD-acireductone complex is limited and possible reaction mechanisms are still under debate. The results of joint experimental and computational studies undertaken to advance knowledge about ARD are reported. The crystal structure of an ARD from Homo sapiens was determined with selenomethionine. EPR spectroscopy suggested that binding acireductone triggers one protein residue to dissociate from Fe2+ , which allows NO (and presumably O2 ) to bind directly to the metal. Mössbauer spectroscopic data (interpreted with the aid of DFT calculations) was consistent with bidentate binding of acireductone to Fe2+ and concomitant dissociation of His88 from the metal. Major features of Fe vibrational spectra obtained for the native enzyme and upon addition of acireductone were reproduced by QM/MM calculations for the proposed models. A computational (QM/MM) study of the reaction mechanisms suggests that Fe2+ promotes O-O bond homolysis, which elicits cleavage of the C1-C2 bond of the substrate. Higher M3+ /M2+ redox potentials of other divalent metals do not support this pathway, and instead the reaction proceeds similarly to the key reaction step in the quercetin 2,3-dioxygenase mechanism.

Rapid identification of tuberculosis epididymo-orchitis by INNO-LiPA Rif TB and QuantiFERON-TB Gold In Tube tests: case report.

Diagnosis of extrapulmonary tuberculosis (TB) is often missed or delayed because of nonspecific clinical and laboratory findings. Novel detection methods, such as polymerase chain reaction and QuantiFERON-TB Gold In Tube, can aid in the diagnosis of active extrapulmonary TB. Here, we demonstrate a case of epididymo-orchitis as the sole presentation of TB in a 32-year-old man.