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.
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 Fe , which allows NO (and presumably O ) to bind directly to the metal. Mössbauer spectroscopic data (interpreted with the aid of DFT calculations) was consistent with bidentate binding of acireductone to Fe 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 Fe promotes O-O bond homolysis, which elicits cleavage of the C1-C2 bond of the substrate. Higher M /M 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.
Most of drugs belong to substances produced by plants and called with a common name as alkaloids. It is a group of organic compounds with one or more nitrogen atoms which are mostly alkaline. To one of the most popular alkaloids morphine or codeine can be counted; both have similar chemical properties. Morphine is mainly used as a painkiller in heroin addiction or as a counter the effects of opioid overdose. Codeine also can be analgesic but causes the disappearance of feeling hungry [1]. Both, morphine and codeine, belong to alkaloids which are produced in opium poppy (Papaver somniferum) at the morphine biosynthesis pathway [2]. The biosynthesis pathways for morphine and berberine engage 2-oxoglutarate dependent dioxygenases (ODD) [3].Presented project concerns production, crystallization, catalytic characterization and engineering of plant enzymes belonging to the superfamily of 2-oxoglutarate dependent dioxygenases. Three enzymes: codeine O-demethylase (CODM), thebaine 6-O-demethylase (T6ODM) and protopine O-dealkylase (PODA) are under investigation. Two of them, CODM and T6ODM, catalyze penultimate and final steps in morphine biosynthesis, while PODA can catalyze the O,O-demethylenation of methylenedioxy bridges on protopine and on protoberberine alkaloids. All three enzymes can catalyze reactions for a relatively wide range of alkaloids as a substrate [2].Number of expression and purification experiments were performed so far, including optimization of different bacterial hosts systems, culture conditions and purification protocols. As a result, active and stable enzymes were obtained. Results of biochemical experiments will be presented.
The aim of this brief review is to provide a roadmap for beginning crystallographers who have little or no experience in structural biology and yet are keen to produce protein crystals and analyze their 3D structures to understand their biological roles. To achieve this goal it is crucial to perform crystallization, structure determination, visualization and analysis of the protein’s structural features related to its biological function. Keeping that objective in mind, tips presented herein cover the most important steps in a crystallographic endeavor and present a selection of databases and software which can aid and accelerate the whole process. We hope that this short overview will help novices coming from different disciplines to navigate a protein crystallography project and, hopefully, allow avoiding some costly mistakes, even though being a crystallographer means learning by trial and error.
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