The biological effects of polyphenolic ellagitannins are mediated by their intestinal metabolites, urolithins. This study investigated redox properties of urolithins A and B using ORAC assay, three cell-based assays, copper-initiated pro-oxidant activity (CIPA) assay, and cyclic voltammetry. Urolithins were strong antioxidants in the ORAC assay, but mostly pro-oxidants in cell-based assays, although urolithin A was an antioxidant in cell culture medium. Parent compound ellagic acid was a strong extracellular antioxidant, but showed no response in the intracellular assay. The CIPA assay confirmed the pro-oxidant activity of ellagitannin metabolites. In the cell proliferation assay, urolithins but not ellagic acid decreased growth and metabolism of HepG2 liver cells. In cyclic voltammetry, the oxidation of urolithin A was partly reversible, but that of urolithin B was irreversible. These results illustrate how strongly measured redox properties depend on the employed assay system and conditions and emphasize the importance of studying pro-oxidant and antioxidant activities in parallel.

Lactate oxidases belong to a group of FMN-dependent enzymes and they catalyze a conversion of lactate to pyruvate with a release of hydrogen peroxide. Hydrogen peroxide is also utilized as a read out in biosensors to quantitate lactate levels in biological samples. Aerococcus viridans lactate oxidase is the best characterized lactate oxidase and our knowledge of lactate oxidases relies largely to studies conducted with that particular enzyme. Pediococcus acidilactici lactate oxidase is also commercially available for e.g. lactate measurements, but this enzyme has not been characterized in detail before. Here we report structural characterization of the recombinant enzyme and its co-factor dependent oligomerization. The crystal structures revealed two distinct conformations in the loop closing the active site, consistent with previous biochemical studies implicating the role of loop in catalysis. Despite the structural conservation of active site residues, we were not able to detect either oxidase or monooxygenase activity when L-lactate was used as a substrate. Pediococcus acidilactici lactate oxidase is therefore an example of a misannotation of an FMNdependent enzyme, which catalyzes likely a so far unknown oxidation reaction.

13Lactate oxidases belong to a group of FMN-dependent enzymes and they catalyze a conversion 14 of lactate to pyruvate with a release of hydrogen peroxide. Hydrogen peroxide is also utilized 15 as a read out in biosensors to quantitate lactate levels in biological samples. Aerococcus 16 viridans lactate oxidase is the best characterized lactate oxidase and our knowledge of lactate 17 oxidases relies largely to studies conducted with that particular enzyme. Pediococcus 18 acidilactici lactate oxidase is also commercially available for e.g. lactate measurements, but 19 this enzyme has not been characterized before in detail. Here we report structural 20 characterization of the recombinant enzyme and its co-factor dependent oligomerization. The 21 crystal structures revealed two distinct conformations in the loop closing the active site, 22 consistent with previous biochemical studies implicating the role of loop in catalysis. Despite 23 the structural conservation of active site residues when compared to Aerococcus viridans 24 lactate oxidase we were not able to detect either oxidase or monooxygenase activity when L-25 lactate or other potential alpha hydroxyl acids were used as a substrate. Pediococcus 26 acidilactici lactate oxidase is therefore an example of a misannotation of an FMN-dependent 27 enzyme, which catalyzes likely a so far unknown oxidation reaction. 28 3 29 4 53 Pediococcus acidilactici proteome does not use lactate as a substrate. We describe here 54 structural and biophysical studies of PaLCTO, which revealed FMN-dependent folding and 55 oligomerization of the enzyme. 56 57 Materials and methods 58 Cloning and site directed mutagenesis 59 Pediococcus acidilactici proteome wide search for keywords lactate, lactate oxidase, α-60 hydroxy acid in Uniprot database gave only one hit that matched with AvLCTO. Similarly, 61 BLASTp search with AvLCTO against P. acidilactici DSM20284 revealed the same hit 62 annotated as putative L-lactate oxidase (Uniprot E0NE46). Genomic DNA of Pediococcus 63 acidilactici (DSM 20284) and Aerococcus viridans (DSM 20340) was obtained from DSMZ, 64 Germany. Gene encoding PaLCTO was cloned into pNH-TrxT (Structural Genomics 65 Consortium) vector using SLIC cloning. The vector encodes for an N-terminal thioredoxin tag 66 with a cleavable TEV protease recognition site. A94G mutant was obtained using standard site-67 directed mutagenesis protocol. All clones were verified using Sanger's dideoxy sequencing. 68 Untagged PaLCTO and AvLCTO were recombinantly expressed from pNIC-CH vector with a 69 stop codon added immediately after the native sequence. Commercial PaLCTO was purchased 70 from Sigma (catalog number LO638, lots STBG2905V and STBF3223V).71 Protein expression and purification 72 Proteins were expressed using BL21 (DE3). Overnight culture was inoculated into terrific broth 73 auto-induction media supplemented with 8 g/L glycerol and 50 μg/ml kanamycin. Cells were 74 grown at 37°C until OD 600 reached 1. The temperature was reduced to 18°C for overnight for 5 75 protein e...

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