Vanillate is converted to protocatechuate by an O-demethylase consisting of VanA and VanB in Streptomyces sp. NL15-2K. In this study, vanillate demethylase from this strain was functionally expressed in Escherichia coli, and its substrate range for vanillate analogs was determined by an in vivo assay using recombinant whole cells. Among aromatic methyl ethers, vanillate, syringate, m-anisate, and veratrate were good substrates, whereas ferulate, vanillin, and guaiacol were not recognized by Streptomyces vanillate demethylase. After vanillate, 4-hydroxy-3-methylbenzoate was a better substrate than m-anisate and veratrate, and the 3-methyl group was efficiently oxidized to a hydroxymethyl group. These observations suggest that the combination of a carboxyl group on the benzene ring and a hydroxyl group in the para-position relative to the carboxyl group may be preferable for substrate recognition by the enzyme.1 H-NMR analysis showed that the demethylation product of veratrate was isovanillate rather than vanillate. Therefore, it was concluded that O-demethylation of veratrate by Streptomyces vanillate demethylase occurred only at the meta-position relative to the carboxyl group. Key words vanillate demethylase; Streptomyces; expression; substrate rangeLignin is a major component of plant biomass, and partial decay of lignin yields numerous aromatic compounds, such as vanillin and catechol, that have many applications in the cosmetics, food, pharmaceutical, and chemical industries. To develop a bioconversion system for lignin-related aromatic compounds, we have focused on the enzymology and genetics of their metabolic pathways in bacteria. In many bacteria, most lignin-related aromatic compounds are degraded to protocatechuate and further broken down via specific ring cleavage pathways. We previously isolated Streptomyces sp. NL15-2K, which degrades various lignin-related aromatic compounds, from forest soil, and have studied the metabolic pathways in this strain. 1-4)Vanillate demethylase is responsible for the conversion of vanillate to protocatechuate in the degradation of lignin-related aromatic compounds in bacteria. This enzyme from Streptomyces sp. NL15-2K is a two-component monooxygenase (class I) consisting of oxygenase and reductase components encoded by vanA and vanB, respectively, 2) and is assumed to convert vanillate to protocatechuate via hydroxylation of the O-methyl group, thereby forming an unstable hemiacetal that spontaneously decomposes into protocatechuate and formaldehyde. 5,6) The Streptomyces vanA (1071 bp) and vanB (936 bp) genes are organized in a cluster and co-transcribed from a putative promoter that is located approximately 61 bp upstream of the initiation codon ATG in vanA.2) The enzymatic characteristics of vanillate demethylases from Pseudomonas spp. and Acinetobacter sp. have been determined in studies using cell-free extracts or recombinant Escherichia coli cells transformed with the vanillate demethylase gene.7-9) Vanillate demethylase from P. testosteroni has a broad substrate...