Identification in Marinomonas mediterranea of a novel quinoprotein with glycine oxidase activity

Abstract

A novel enzyme with lysine-epsilon oxidase activity was previously described in the marine bacterium Marinomonas mediterranea. This enzyme differs from other l-amino acid oxidases in not being a flavoprotein but containing a quinone cofactor. It is encoded by an operon with two genes lodA and lodB. The first one codes for the oxidase, while the second one encodes a protein required for the expression of the former. Genome sequencing of M. mediterranea has revealed that it contains two additional operons encoding proteins with sequence similarity to LodA. In this study, it is shown that the product of one of such genes, Marme_1655, encodes a protein with glycine oxidase activity. This activity shows important differences in terms of substrate range and sensitivity to inhibitors to other glycine oxidases previously described which are flavoproteins synthesized by Bacillus. The results presented in this study indicate that the products of the genes with different degrees of similarity to lodA detected in bacterial genomes could constitute a reservoir of different oxidases.

Keywords: Glycine oxidase; Marinomonas mediterranea; hydrogen peroxide; quinoprotein..

Figure 1

Antimicrobial and oxidase activity in Marinomonas mediterraneaLD (ΔlodAB) supernatants. (A) Antibiograms in LB medium against Escherichia coli UM202. LD, disk loaded with 20 μL of 10× concentrated supernatant; CAT, disk loaded with 20 μL of catalase 10 mg/mL. (B) Oxidase activity against the proteinogenic amino acids at a concentration of 2 mmol/L measured using the fluorimetric assay for hydrogen peroxide detection. Activity is expressed as relative fluorescence units (RFU) per min and mg of protein.

Figure 2

Sensitivity of Marinomonas mediterraneaGOX (filled bars) to quinoprotein inhibitors. Controls: lysine-α-oxidase, a flavoprotein (empty bars), Marinomonas mediterranea LodA, a quinoprotein (gray bars), and horseradish peroxidase (HPR, stripped bars). The effect of remaining phenylhydrazine and methylhydrazine on HRP was determined by using 10 μmol/L H₂O₂ as substrate for the reaction.

Figure 3

Identification of Marme_1655 as the gene encoding Gox. 100× concentrated supernatants of Marinomonas mediterraneaLD (ΔlodAB) were run in SDS-PAGE under nondenaturing conditions. The gel was sliced in different fragments. The fragment between, approximately, 130–170 kDa showed antimicrobial activity, glycine oxidase activity, and several peptides matching the product of Marme_1655.

Figure 4

Sequence analysis of the product of Marme_1655. Bold letters indicate peptides detected after trypsin digestion and HPLC-MS/MS analysis. Highlighted sequences indicate conserved regions detected also in LodA and similar proteins (Lucas-Elio et al. 2006). Underlined sequence corresponds to the signal peptide with the twin-arginine motif (capital letters) predicted according to the TatP 1.0 server (Bendtsen et al. 2005).

Figure 5

Genome region of Marinomonas mediterranea around gene Marme_1655.

Figure 6

Glycine oxidase activity in supernatants of different Marinomonas mediterranea strains. The strains were grown for 48 h in MNGL. GOX activity is expressed as relative fluorescence units (RFU) per min and mg of protein.