Effect of Cu(II) and Conserved Copper Binding Sites on Multicopper Oxidase CopA and Characterization of the BioMnOx

The microbial manganese removal process is believed to be the catalytic oxidation of Mn(II) by manganese oxidase. In this study, the multicopper oxidase CopA was purified and found to have high manganese oxidation activity in vitro and Cu(II) can significantly enhance its manganese oxidation activity. The gene site-directed mutagenesis was used to mutate four conserved copper binding sites of CopA and then obtain four mutant strains. The manganese removal efficiency of the four strains was determined to find that H120 is the catalytic active site of the CopA. Protein modification analysis of CopA obtained under different conditions by mass spectrometry revealed that the loss of Cu(Ⅱ) and the mutation of the conserved copper binding site H120 resulted in the loss of modification of ethoxyformyl and quinone, the number of modifications was reduced and the position of modification was changed, eventually causing a decrease in protein activity. It reveals that Cu(II) and H120 play an indispensable role in the manganese oxidation of the multicopper oxidase CopA. The Mn valence state of BioMnOx was analyzed by XPS, finding that both the strain-mediated product and the CopA-mediated product were composed of MnO2 and Mn3O4 and the average valence of Mn is 3.2.

Monapinone Coupling Enzyme Produces Non-Natural Heterodimers

The monapinone coupling enzyme (MCE), a fungal multicopper oxidase, catalyzes the regioselective C–C coupling between tricyclic monapinone A (the primary substrate) and other monapinones (secondary substrates) to produce atropisomeric biaryl homo- or heterodimers. In this study, mono-, bi- and tricyclic compounds were tested to determine whether they worked as secondary substrates for MCE. Among 14 cyclic compounds, MCE utilized semivioxanthin, YWA1, 1,3-naphthalenediol and flaviolin as secondary substrates to produce non-natural heterodimers. The atropisomeric biaryl heterodimers produced by MCE from monapinone A and semivioxanthin were isolated, and their structures were elucidated by NMR and MS. These findings indicate that MCE recognizes bi- and tricyclic compounds with a 1,3-dihydroxy or 1-hydroxy-3-methoxy benzene ring as a secondary substrate.

Biochemical properties of CumA multicopper oxidase from plant pathogen, Pseudomonas syringae

Multicopper oxidases have a wide range of substrate specificity to be involved in various physiological reactions. Pseudomonas syringae, a plant pathogenic bacterium, has a multicopper oxidase, CumA. Multicopper oxidases have ability to degrade plant cell wall component, lignin. Once P. syringae enter apoplast and colonize, they start to disrupt plant immunity. Therefore, deeper understanding of multicopper oxidases from plant pathogens, help to invent measures to prevent invasion into plant cell, which bring agricultural benefits. Several biochemical studies have reported lower activity of CumA compared with other multicopper oxidase called CotA. However, the mechanisms underlying the difference in activity have not yet been revealed. In order to acquire insight into them, we conducted a biophysical characterization of PsCumA. Our results show that PsCumA has weak type I copper EPR signal, which is essential for oxidation activity. We propose that difference in the coordination of copper ions may decrease reaction frequency.

Genome analysis of Pseudomonas sp. OF001 and Rubrivivax sp. A210 suggests multicopper oxidases catalyze manganese oxidation required for cylindrospermopsin transformation

Background Cylindrospermopsin is a highly persistent cyanobacterial secondary metabolite toxic to humans and other living organisms. Strain OF001 and A210 are manganese-oxidizing bacteria (MOB) able to transform cylindrospermopsin during the oxidation of Mn2+. So far, the enzymes involved in manganese oxidation in strain OF001 and A210 are unknown. Therefore, we analyze the genomes of two cylindrospermopsin-transforming MOB, Pseudomonas sp. OF001 and Rubrivivax sp. A210, to identify enzymes that could catalyze the oxidation of Mn2+. We also investigated specific metabolic features related to pollutant degradation and explored the metabolic potential of these two MOB with respect to the role they may play in biotechnological applications and/or in the environment. Results Strain OF001 encodes two multicopper oxidases and one haem peroxidase potentially involved in Mn2+ oxidation, with a high similarity to manganese-oxidizing enzymes described for Pseudomonas putida GB-1 (80, 83 and 42% respectively). Strain A210 encodes one multicopper oxidase potentially involved in Mn2+ oxidation, with a high similarity (59%) to the manganese-oxidizing multicopper oxidase in Leptothrix discophora SS-1. Strain OF001 and A210 have genes that might confer them the ability to remove aromatic compounds via the catechol meta- and ortho-cleavage pathway, respectively. Based on the genomic content, both strains may grow over a wide range of O2 concentrations, including microaerophilic conditions, fix nitrogen, and reduce nitrate and sulfate in an assimilatory fashion. Moreover, the strain A210 encodes genes which may convey the ability to reduce nitrate in a dissimilatory manner, and fix carbon via the Calvin cycle. Both MOB encode CRISPR-Cas systems, several predicted genomic islands, and phage proteins, which likely contribute to their genome plasticity. Conclusions The genomes of Pseudomonas sp. OF001 and Rubrivivax sp. A210 encode sequences with high similarity to already described MCOs which may catalyze manganese oxidation required for cylindrospermopsin transformation. Furthermore, the analysis of the general metabolism of two MOB strains may contribute to a better understanding of the niches of cylindrospermopsin-removing MOB in natural habitats and their implementation in biotechnological applications to treat water.