title: Three-Component O-Demethylase System Essential for Catabolism of a Lignin-Derived Biphenyl Compound in Sphingobium sp. Strain SYK-6.
abstract: Sphingobium sp. strain SYK-6 is able to assimilate lignin-derived biaryls, including a biphenyl compound, 5,5'-dehydrodivanillate (DDVA). Previously, ligXa (SLG_07770), which is similar to the gene encoding oxygenase components of Rieske-type nonheme iron aromatic-ring-hydroxylating oxygenases, was identified to be essential for the conversion of DDVA; however, the genes encoding electron transfer components remained unknown. Disruption of putative electron transfer component genes scattered through the SYK-6 genome indicated that SLG_08500 and SLG_21200, which showed approximately 60% amino acid sequence identities with ferredoxin and ferredoxin reductase of dicamba O-demethylase, were essential for the normal growth of SYK-6 on DDVA. LigXa and the gene products of SLG_08500 (LigXc) and SLG_21200 (LigXd) were purified and were estimated to be a trimer, a monomer, and a monomer, respectively. LigXd contains FAD as the prosthetic group and showed much higher reductase activity toward 2,6-dichlorophenolindophenol with NADH than with NADPH. A mixture of purified LigXa, LigXc, and LigXd converted DDVA into 2,2',3-trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl in the presence of NADH, indicating that DDVA O-demethylase is a three-component monooxygenase. This enzyme requires Fe(II) for its activity and is highly specific for DDVA, with a Km value of 63.5 μM and kcat of 6.1 s(-1). Genome searches in six other sphingomonads revealed genes similar to ligXc and ligXd (>58% amino acid sequence identities) with a limited number of electron transfer component genes, yet a number of diverse oxygenase component genes were found. This fact implies that these few electron transfer components are able to interact with numerous oxygenase components and the conserved LigXc and LigXd orthologs are important in sphingomonads.
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Taichi
authors.initials: T
authors.lastname: Yoshikata
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Suzuki
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Naofumi
authors.initials: N
authors.lastname: Kamimura
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Namiki
authors.affiliation: Forestry and Forest Products Research Institute, Tsukuba, Ibaraki, Japan.
authors.forename: Shojiro
authors.initials: S
authors.lastname: Hishiyama
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Takuma
authors.initials: T
authors.lastname: Araki
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kasai
authors.affiliation: Forestry and Forest Products Research Institute, Tsukuba, Ibaraki, Japan.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Otsuka
authors.affiliation: Forestry and Forest Products Research Institute, Tsukuba, Ibaraki, Japan.
authors.forename: Masaya
authors.initials: M
authors.lastname: Nakamura
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Masao
authors.initials: M
authors.lastname: Fukuda
authors.affiliation: College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Katayama
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan emasai@vos.nagaokaut.ac.jp.
authors.forename: Eiji
authors.initials: E
authors.lastname: Masai
mesh_terms: D001711:Biotransformation; D001713:Biphenyl Compounds / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010090:Oxidoreductases, O-Demethylating / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D055503:Protein Multimerization; D042301:Sphingomonadaceae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001713:Biphenyl Compounds; D009243:NAD; D006899:Mixed Function Oxygenases; D010090:Oxidoreductases, O-Demethylating