Expression and Substrate Range of <i>Streptomyces</i> Vanillate Demethylase

Abstract

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...

Exploring the Selective Demethylation of Aryl Methyl Ethers with a Pseudomonas Rieske Monooxygenase

“…No activity was detected; this suggests the requirement for a carboxylic moiety for substrate recognition. This result is in accordance with previous in vivo studies on Acinetobacter , Streptomyces , and Pseudomonas fluorescens vanillate demethylases . The presence of longer aliphatic chains in bulkier compounds was unfavorable as well: homovanillic acid, ferulic acid, dihydroferulic acid, sinapyl acid, and vanilpyruvic acid were not accepted and no product formation or substrate consumption was observed.…”

“…This result distinguishes Ps (HR199)VanA from both Ac VanA and St VanA. The first cannot convert 7 a , whereas St VanA is less specific and generates a mixture of 8 a and gallate …”

“…One example of such a two‐component system is the O‐demethylase from Streptomyces ( St VanA) partially characterized by Nishimura and co‐workers . They explored the substrate scope of St VanA after recombinant protein expression, using whole‐cell bioconversions with E. coli .…”

Cleavage of Methyl Ethers by O-Desmethylangolensin-Producing Clostridium Strain SY8519

“…Therefore, we did not observe selectivity for demethylation between the 3-O-and 4-O-methyl moieties of 1. From the results, however, we conclude that strain SY8519 has selectively for the demethylation as like reported enzymes [11,12]. The different rate of demethylation produced 3 as a metabolite from 1 and 2 (Figure 2a).…”

Anaerobic degradation of syringic acid by an adapted strain ofRhodopseudomonas palustris

“…strain HR199 (47). In addition, a VanAB homologue in a Streptomyces strain has the reported ability to demethylate syringic acid as well as other aromatic compounds (48).…”