Manganese peroxidases (MnP) from the white-rot fungi Phanerochaete chrysosporium catalyse the oxidation of Mn2+ to Mn3+, a strong oxidizer able to oxidize a wide variety of organic compounds. Different approaches have been used to unravel the enzymatic properties and potential applications of MnP. However, these efforts have been hampered by the limited production of native MnP by fungi. Heterologous expression of MnP has been achieved in both eukaryotic and prokaryotic expression systems, although with limited production and many disadvantages in the process. Here we described a novel molecular approach for the expression and purification of manganese peroxidase isoform 1 (MnP1) from P. chrysosporium using an E. coli-expression system. The proposed strategy involved the codon optimization and chemical synthesis of the MnP1 gene for optimised expression in the E. coli T7 shuffle host. Recombinant MnP1 (rMnP1) was expressed as a fusion protein, which was recovered from solubilised inclusion bodies. rMnP1 was purified from the fusion protein using intein-based protein purification techniques and a one-step affinity chromatography. The designated strategy allowed production of an active enzyme able to oxidize guaiacol or Mn2+.

The opportunistic human pathogen Pseudomonas aeruginosa exhibits great resistance to antibiotics; so, new therapeutic agents are urgently needed. Since polyamines levels are incremented in infected tissues, we explored whether the formation of a toxic aldehyde in polyamines degradation can be exploited in combating infection. We cloned the gene encoding the only aminoaldehyde dehydrogenase involved in P. aeruginosa polyamines‐degradation routes, PaPauC, overexpressed this enzyme, and found that it oxidizes 3‐aminopropionaldehyde (APAL) and 3‐glutamyl‐3‐aminopropionaldehyde (GluAPAL) − produced in spermine (Spm), spermidine (Spd), and diaminopropane (Dap) degradation, as well as 4‐aminobutyraldehyde (ABAL) and 4‐glutamyl‐4‐aminobutyraldehyde (GluABAL) − formed in putrescine (Put) degradation. As the catalytic efficiency of PaPauC with APAL was 30‐times lower than with GluAPAL, and GluAPAL is predominantly formed, APAL will be poorly oxidized ‘in vivo’. We found polyamines‐induced increases in the PaPauC activity of cell crude‐extracts and in the expression of the PapauC gene that were diminished by glucose. Spm, Spd, or Dap, but not Put, were toxic to P. aeruginosa even in the presence of other carbon and nitrogen sources, particularly to a strain with the PapauC gene disrupted. APAL, but not GluAPAL, was highly toxic even to wild‐type cells, suggesting that its accumulation, particularly in the absence of, or low, PaPauC activity is responsible for the toxicity of Spm, Spd, and Dap. Our results shed light on the toxicity mechanism of these three polyamines and strongly support the critical role of PaPauC in this toxicity. Thus, PaPauC emerges as a novel potential drug target whose inhibition might help in combating infection by this important pathogen.

Few land plants can synthesize and accumulate the osmoprotectant glycine betaine (GB) even though this metabolic trait has major adaptive importance given the prevalence of drought, hypersaline soils or cold. GB is synthesized from choline in two reactions catalyzed by choline monooxygenases (CMOs) and enzymes of the family 10 of aldehyde dehydrogenases (ALDH10s) that gained betaine aldehyde dehydrogenase activity (BADH). Homolog genes encoding CMO and ALDH10 enzymes are present in all known land plant genomes, but since GB-non-accumulators plants lack the BADH-type ALDH10 isozyme, they would be expected to also lack the CMO activity to avoid accumulation of the toxic betaine aldehyde. To explore CMOs substrate specificity, we performed amino acid sequence alignments, phylogenetic analysis, homology modeling and docking simulations. We found that plant CMOs form a monophyletic subfamily within the Rieske/mononuclear non-heme oxygenases family with two clades: CMO1 and CMO2, the latter diverging from CMO1 after gene duplication. CMO1 enzymes are present in all plants; CMO2s only in the Amaranthaceae high-GB-accumulators plants. CMO2s, and particularly their mononuclear non-heme iron domain where the active site is located, evolved at a faster rate than CMO1s, which suggests positive selection. The homology model and docking simulations of the spinach CMO2 enzyme showed at the active site three aromatic residues forming a box with which the trimethylammonium group of choline could interact through cation-π interactions, and a glutamate, which also may interact with the trimethylammonium group through a charge-charge interaction. The aromatic box and the carboxylate have been shown to be critical for choline binding in other proteins. Interestingly, these residues are conserved in CMO2 proteins but not in CMO1 proteins, where two of these aromatic residues are leucine and the glutamate is asparagine. These findings reinforce our proposal that the CMO1s physiological substrate is not choline but a still unknown metabolite.

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Las oxigenasas Rieske/mononuclear son un grupo de metaloenzimas que catalizan la oxidación de una variedad de compuestos, destaca su participación en la degradación de compuestos xenobióticos contaminantes; estas enzimas también participan en la biosíntesis de algunos compuestos de interés comercial. Poseen una amplia especificidad por el sustrato, convirtiéndolas en un grupo de enzimas con un alto potencial de aplicación en procesos biotecnológicos que hasta el momento no ha sido explotado. La presente revisión aborda aspectos generales acerca de la función y estructura de este importante grupo de enzimas.