Identification of a Phylogenetically Divergent Vanillate O-Demethylase from Rhodococcus ruber R1 Supporting Growth on Meta-Methoxylated Aromatic Acids

Rieske-type two-component vanillate O-demethylases (VanODs) catalyze conversion of the lignin-derived monomer vanillate into protocatechuate in several bacterial species. Currently, VanODs have received attention because of the demand of effective lignin valorization technologies, since these enzymes own the potential to catalyze methoxy group demethylation of distinct lignin monomers. In this work, we identified a phylogenetically divergent VanOD from Rhodococcus ruber R1, only distantly related to previously described homologues and whose presence, along with a 3-hydroxybenzoate/gentisate pathway, correlated with the ability to grow on other meta-methoxylated aromatics, such as 3-methoxybenzoate and 5-methoxysalicylate. The complementation of catabolic abilities by heterologous expression in a host strain unable to grow on vanillate, and subsequent resting cell assays, suggest that the vanAB genes of R1 strain encode a proficient VanOD acting on different vanillate-like substrates; and also revealed that a methoxy group in the meta position and a carboxylic acid moiety in the aromatic ring are key for substrate recognition. Phylogenetic analysis of the oxygenase subunit of bacterial VanODs revealed three divergent groups constituted by homologues found in Proteobacteria (Type I), Actinobacteria (Type II), or Proteobacteria/Actinobacteria (Type III) in which the R1 VanOD is placed. These results suggest that VanOD from R1 strain, and its type III homologues, expand the range of methoxylated aromatics used as substrates by bacteria.