Discovery and characterization of l-DOPA 2,3-dioxygenase from Streptomyces hygroscopicus jingganensis.

The largest natural reservoir of untapped carbon can be found in the cell-wall strengthening, plant woody-tissue polymer, lignin - a polymer of catechols or 1,2-dihydroxybenzene monomers. The catecholic carbon of lignin could be valorized into feedstocks and natural products by way of catabolic and biosynthetic transformations, including the oxygen-dependent cleavage reaction of extradiol dioxygenase (EDX) enzymes. The EDX l-DOPA 2,3-dioxygenase was first discovered as part of a biosynthetic gene cluster to the natural product antibiotic, lincomycin, and also contributes to the biosyntheses of anthramycin, sibiromycin, tomaymycin, porothramycin and hormaomycin. Using these l-DOPA 2,3-dioxygenases as a starting point, we searched sequence space in order to identify new sources of dioxygenase driven natural product diversity. A "vicinal-oxygen-chelate (VOC) family protein" from Streptomyces hygroscopicus jingganensis was identified using bioinformatic methods and biochemically investigated for dioxygenase activity against a suite of natural and synthetic catechols. Steady-state oxygen consumption assays were used to screen and identify substrates, and a steady-state kinetic model of oxygen consumption was developed to evaluate activity of the S. hygroscopicus jingganensis VOC-family-protein with respect to activity of l-DOPA 2,3-dioxygenases from Streptomyces lincolnensis and Streptomyces sclerotialus. Lastly, these data were integrated with steady-state kinetic methods to observe the formation of the EDX cleavage product with UV-visible spectroscopy. The genomic context and enzymatic activity of the S. hygroscopicus jingganensis VOC family protein are consistent with a l-DOPA 2,3-dioxygenase contained within a cryptic biosynthetic pathway.

Measurement of 6-cyanodopamine, 6-nitrodopa, 6-nitrodopamine and 6-nitroadrenaline by LC-MS/MS in Krebs-Henseleit solution. Assessment of basal release from rabbit isolated right atrium and ventricles.

This study presents the validation of a sensitive method for the determination of 6-nitrodopa, 6-nitrodopamine, 6-nitroadrenaline and 6-cyanodopamine in Krebs-Henseleit solution by LC-MS/MS with ESI+ . HRMS was used to precisely characterize the structures of the fragment ions. The method was applied to investigate the catecholamine basal release from rabbit isolated atria and ventricles. The atria and ventricles were suspended separately in a 5 ml organ bath containing Krebs-Henseleit solution with ascorbic acid (3 mM), gassed (95%O2 /5%CO2 ) at 37°C for 30 min. Strata-X 33 µm SPE cartridges were employed for the extraction of the catecholamines and the internal standard 6-nitrodopamine-d4 . The catecholamines were separated employing a 150 × 3 mm Shim-pack GIST C18-AQ (3 mm particle size), placed in an oven at 40°C and perfused by 65% of mobile phase A (MeCN/H2 O; 90/10, v/v) + 0.4% CH3 COOH and 35% mobile phase B (deionized H2 O) + 0.2% CH2 O2 at 320 µl/min in isocratic mode. The method was linear at 0.1-20 ng/ml. The method was used to identify for the first-time basal release of the three nitrocatecholamines mentioned above and a member of a novel class of catecholamines, the cyanocatecholamines.

L-DOPA Dioxygenase Activity on 6-Substituted Dopamine Analogues.

Dioxygenase enzymes are essential protein catalysts for the breakdown of catecholic rings, structural components of plant woody tissue. This powerful chemistry is used in nature to make antibiotics and other bioactive materials or degrade plant material, but we have a limited understanding of the breadth and depth of substrate space for these potent catalysts. Here we report steady-state and pre-steady-state kinetic analysis of dopamine derivatives substituted at the 6-position as substrates of L-DOPA dioxygenase, and an analysis of that activity as a function of the electron-withdrawing nature of the substituent. Steady-state and pre-steady-state kinetic data demonstrate the dopamines are impaired in binding and catalysis with respect to the cosubstrate molecular oxygen, which likely afforded spectroscopic observation of an early reaction intermediate, the semiquinone of dopamine. The reaction pathway of dopamine in the pre-steady state is consistent with a nonproductive mode of binding of oxygen at the active site. Despite these limitations, L-DOPA dioxygenase is capable of binding all of the dopamine derivatives and catalyzing multiple turnovers of ring cleavage for dopamine, 6-bromodopamine, 6-carboxydopamine, and 6-cyanodopamine. 6-Nitrodopamine was a single-turnover substrate. The variety of substrates accepted by the enzyme is consistent with an interplay of factors, including the capacity of the active site to bind large, negatively charged groups at the 6-position and the overall oxidizability of each catecholamine, and is indicative of the utility of extradiol cleavage in semisynthetic and bioremediation applications.