Membrane-Associated Glucose-Methanol-Choline Oxidoreductase Family Enzymes PhcC and PhcD Are Essential for Enantioselective Catabolism of Dehydrodiconiferyl Alcohol

Abstract

e Sphingobium sp. strain SYK-6 is able to degrade various lignin-derived biaryls, including a phenylcoumaran-type compound, dehydrodiconiferyl alcohol (DCA). In SYK-6 cells, the alcohol group of the B-ring side chain of DCA is initially oxidized to the carboxyl group to generate 3-(2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-7-methoxy-2,3-dihydrobenzofuran-5-yl) acrylic acid (DCA-C). Next, the alcohol group of the A-ring side chain of DCA-C is oxidized to the carboxyl group, and then the resulting metabolite is catabolized through vanillin and 5-formylferulate. In this study, the genes involved in the conversion of DCA-C were identified and characterized. The DCA-C oxidation activities in SYK-6 were enhanced in the presence of flavin adenine dinucleotide and an artificial electron acceptor and were induced ca. 1.6-fold when the cells were grown with DCA. Based on these observations, SLG_09480 (phcC) and SLG_09500 (phcD), encoding glucose-methanol-choline oxidoreductase family proteins, were presumed to encode DCA-C oxidases. Analyses of phcC and phcD mutants indicated that PhcC and PhcD are essential for the conversion of (؉)-DCA-C and (؊)-DCA-C, respectively. When phcC and phcD were expressed in SYK-6 and Escherichia coli, the gene products were mainly observed in their membrane fractions. The membrane fractions of E. coli that expressed phcC and phcD catalyzed the specific conversion of DCA-C into the corresponding carboxyl derivatives. In the oxidation of DCA-C, PhcC and PhcD effectively utilized ubiquinone derivatives as electron acceptors. Furthermore, the transcription of a putative cytochrome c gene was significantly induced in SYK-6 grown with DCA. The DCA-C oxidation catalyzed by membrane-associated PhcC and PhcD appears to be coupled to the respiratory chain. L ignin, one of the major components of plant cell walls, is a complex phenolic polymer resulting from the oxidative combinatorial coupling of 4-hydroxycinnamyl alcohols (1). Although lignin has various intermolecular linkages between phenylpropane units and contains a number of asymmetric carbons, it is considered to be optically inactive, implying the racemic nature of the lignin backbone (2-4). In nature, lignin is initially decomposed by phenol oxidases such as lignin peroxidase, manganese peroxidase, versatile peroxidase, and laccase secreted by white rot fungi (5-7). Recently, dye-decolorizing peroxidases (Dyp) of Rhodococcus (8) and Amycolatopsis (9) and small laccase of Streptomyces (10) were characterized, and these enzymes have been implicated as being involved in lignin degradation. In addition, bacteria play key roles in the degradation and mineralization of low-molecular-weight aromatic compounds derived from lignin (11, 12). Since fragmented oligomers from lignin consist of stereoisomers that contain various types of intermolecular linkages between phenylpropane units, catabolic enzymes necessary for the conversion of such stereoisomers must have evolved in bacteria to fully utilize structurally and stereochemically complicated l...

Two novel decarboxylase genes play a key role in the stereospecific catabolism of dehydrodiconiferyl alcohol in Sphingobium sp. strain SYK‐6

“…PhcF and PhcG enantiospecifically catalyse the decarboxylation of (+)‐DCA‐CC and (–)‐DCA‐CC respectively, into achiral DCA‐S and play an important role in their conversions in SYK‐6. This study together with our previous report (Takahashi et al ., ) demonstrates the whole picture of the bacterial enzyme system for the conversion of enantiomers of phenylcoumaran derivatives into achiral DCA‐S. In addition, this study will contribute to a better understanding of the function of the DUF3237 superfamily enzymes.…”

“…DCA-C, DCA-CC, DCA-S and DCA-AC were prepared as described previously (Takahashi et al, 2014;Takahashi et al, 2015). Vanillic acid, homovanillic acid, homoprotocatechuic acid, vanillylmandelic acid, ferulic acid, sinapinic acid, cinnamic acid, 3-methoxycinnamic acid, 4-methoxycinnamic acid, o-coumaric acid, m-coumaric acid and p-coumaric acid were purchased from Tokyo Chemical Industry or Wako Pure Chemical Industries.…”

“…The (1)-DCA-C oxidase gene phcC was expressed in E. coli as described previously (Takahashi et al, 2015). Crude PhcC (50 lg/ml of protein) was incubated in a reaction mixture containing 400 lM DCA-C, 600 lM FAD, 600 lM 1-methoxy-5methylphenazinium methylsulfate (PMS) and buffer A at 308C for 60 min to identify the peak of (1)-DCA-CC of a racemic DCA-CC preparation separated by chiral-HPLC.…”

“…The DCA catabolic pathways have been determined for Sphingomonas paucimobilis TMY1009 (Habu et al, 1988) and SYK-6, and efforts to identify the enzymes and genes involved in DCA catabolism in SYK-6 have been ongoing in recent years ( Fig. 1) (Takahashi et al, 2014;Takahashi et al, 2015).…”

“…In the oxidation processes to convert DCA into DCA-C, involvement of multiple aryl alcohol dehydrogenases and aldehyde dehydrogenases (ALDHs) were suggested in a previous study (Takahashi et al, 2014). PhcC and PhcD, which belong to the glucose-methanol-choline oxidoreductase family, catalysed the stereospecific conversion of (1)-DCA-C and (-)-DCA-C respectively (Takahashi et al, 2015). The resulting DCA-CL was converted to DCA-CC mainly by NAD 1 -dependent ALDHs although PhcC and PhcD were also able to convert DCA-C to DCA-CC via DCA-CL.…”