title: Novel polyphenol oxidase mined from a metagenome expression library of bovine rumen: biochemical properties, structural analysis, and phylogenetic relationships.
abstract: RL5, a gene coding for a novel polyphenol oxidase, was identified through activity screening of a metagenome expression library from bovine rumen microflora. Characterization of the recombinant protein produced in Escherichia coli revealed a multipotent capacity to oxidize a wide range of substrates (syringaldazine > 2,6-dimethoxyphenol > veratryl alcohol > guaiacol > tetramethylbenzidine > 4-methoxybenzyl alcohol > 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) >> phenol red) over an unusually broad range of pH from 3.5 to 9.0. Apparent Km and kcat values for ABTS, syringaldazine, and 2,6-dimetoxyphenol obtained from steady-state kinetic measurements performed at 40 degrees C, pH 4.5, yielded values of 26, 0.43, and 0.45 microm and 18, 660, and 1175 s(-1), respectively. The Km values for syringaldazine and 2,6-dimetoxyphenol are up to 5 times lower, and the kcat values up to 40 times higher, than values previously reported for this class of enzyme. RL5 is a 4-copper oxidase with oxidation potential values of 745, 400, and 500 mV versus normal hydrogen electrode for the T1, T2, and T3 copper sites. A three-dimensional model of RL5 and site-directed mutants were generated to identify the copper ligands. Bioinformatic analysis of the gene sequence and the sequences and contexts of neighboring genes suggested a tentative phylogenetic assignment to the genus Bacteroides. Kinetic, electrochemical, and EPR analyses provide unequivocal evidence that the hypothetical proteins from Bacteroides thetaiotaomicron and from E. coli, which are closely related to the deduced protein encoded by the RL5 gene, are also multicopper proteins with polyphenol oxidase activity. The present study shows that these three newly characterized enzymes form a new family of functional multicopper oxidases with laccase activity related to conserved hypothetical proteins harboring the domain of unknown function DUF152 and suggests that some other of these proteins may also be laccases.
authors.affiliation: Institute of Catalysis, Consejo Superior de Investigaciones Científicas (CSIC), Cantoblanco, 28049 Madrid, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Beloqui
authors.forename: Marcos
authors.initials: M
authors.lastname: Pita
authors.forename: Julio
authors.initials: J
authors.lastname: Polaina
authors.forename: Arturo
authors.initials: A
authors.lastname: Martínez-Arias
authors.forename: Olga V
authors.initials: OV
authors.lastname: Golyshina
authors.forename: Miren
authors.initials: M
authors.lastname: Zumárraga
authors.forename: Michail M
authors.initials: MM
authors.lastname: Yakimov
authors.forename: Humberto
authors.initials: H
authors.lastname: García-Arellano
authors.forename: Miguel
authors.initials: M
authors.lastname: Alcalde
authors.forename: Víctor M
authors.initials: VM
authors.lastname: Fernández
authors.forename: Kieran
authors.initials: K
authors.lastname: Elborough
authors.forename: José M
authors.initials: JM
authors.lastname: Andreu
authors.forename: Antonio
authors.initials: A
authors.lastname: Ballesteros
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Plou
authors.forename: Kenneth N
authors.initials: KN
authors.lastname: Timmis
authors.forename: Manuel
authors.initials: M
authors.lastname: Ferrer
authors.forename: Peter N
authors.initials: PN
authors.lastname: Golyshin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001439:Bacteroides / Q000201:enzymology*; D004156:Catechol Oxidase / Q000737:chemistry*; D002417:Cattle; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D007422:Intestines / Q000382:microbiology; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid
chemical_list: D004156:Catechol Oxidase