Novel Polyphenol Oxidase Mined from a Metagenome Expression Library of Bovine Rumen

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) Ͼ Ͼ phenol red) over an unusually broad range of pH from 3.5 to 9.0. Apparent K m and k cat 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 M and 18, 660, and 1175 s ؊1 , respectively. The K m values for syringaldazine and 2,6-dimetoxyphenol are up to 5 times lower, and the k cat 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.Laccases are multicopper oxidoreductases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) able to oxidize a wide variety of phenolic and nonphenolic compounds, including industrial dyes, polycyclic aromatic hydrocarbons, pesticides, and alquenes, but also capable of performing polymerization, depolymerization, methylation, and demethylation reactions (Refs.

Microbial Metabolic Potential for Carbon Degradation and Nutrient (Nitrogen and Phosphorus) Acquisition in an Ombrotrophic Peatland

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Latex Clearing Protein—an Oxygenase Cleaving Poly( cis -1,4-Isoprene) Rubber at the cis Double Bonds

“…Due to the inhibition of Lcp1 VH2 activity by the utilized chelating agents with a simultaneous absence of metals other than copper and the results of the applied bathocuproine sulfonate-based spectroscopic assays, and since it is known from the literature that hexahistidine-tagged, purified, copper-binding enzymes do not contain significantly larger amounts of copper than the same proteins that were purified by applying streptavidin tags or for which the hexahistidine tag was removed (43,44), it can be concluded that purified Lcp1 VH2 possesses Cu(II) as a transition factor. The divalent copper ions are known to preferably complex with histidine, serine, threonine, or tyrosine amino acid residues (45).…”

Structure, functionality and tuning up of laccases for lignocellulose and other industrial applications

“…Laccases are produced by bacteria (Beloqui et al, 2006), insects (Dittmer et al, 2004), plants (O'Malley et al, 1993) and fungi (Basidomycota and Ascomycota). Laccases produced by white-rot fungi (Basidiomycota) currently receive particular attention due to their apparent ability to catalyze the modification and depolymerization of lignin and in this way assist in the decomposition of plant material.…”