2024-03-29T05:36:35Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/1637602020-10-23T12:49:35Zcom_10261_131com_10261_2com_10261_86com_10261_1com_10261_101com_10261_5col_10261_384col_10261_339col_10261_354
Novel Polyphenol Oxidase Mined from a Metagenome Expression Library of Bovine Rumen
Beloqui, Ana
Pita, Marcos
Polaina Molina, Julio
Martínez Arias, Arturo
Zumárraga, Miren
García-Arellano, Humberto
Alcalde Galeote, Miguel
Andreu, José Manuel
Ballesteros Olmo, Antonio
Plou Gasca, Francisco José
Ferrer, Manuel
Golyshin, Peter N.
ViaLactiaBiosciences
Federal Ministry of Education and Research (Germany)
European Commission
Ministerio de Educación y Ciencia (España)
Eusko Jaurlaritza
Comunidad de Madrid
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 °C, pH 4.5, yielded values of 26, 0.43, and 0.45 μm 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.
2018-04-18T08:52:17Z
2018-04-18T08:52:17Z
2006-06
artículo
Journal of Biological Chemistry 281(32): 22933-22942 (2006)
0021-9258
http://hdl.handle.net/10261/163760
10.1074/jbc.M600577200
1083-351X
http://dx.doi.org/10.13039/501100000780
http://dx.doi.org/10.13039/501100002347
http://dx.doi.org/10.13039/501100003086
http://dx.doi.org/10.13039/100012818
eng
Publisher's version
http://dx.doi.org/10.1074/jbc.M600577200
Sí
openAccess
American Society for Biochemistry and Molecular Biology