Molecular cloning and functional characterization of a unique multipotent polyphenol oxidase from Marinomonas mediterranea.
Marinomonas mediterranea is a recently isolated melanogenic marine bacterium containing laccase and tyrosinase activities. These activities are due to the expression of two polyphenol oxidases (PPOs), a blue multicopper laccase and an SDS-activated tyrosinase. The gene encoding the first one, herein denominated M. mediterranea PpoA, has been isolated by transposon mutagenesis, cloned and expressed in Escherichia coli. Its predicted amino acid sequence shows the existence of a signal peptide and four copper-binding sites characteristic of the blue multicopper proteins, including all fungal laccases. In addition, two additional putative copper-binding sites near its N-terminus are also present. Recombinant expression in E. coli of this protein clearly demonstrates its multipotent capability, showing both laccase-like and tyrosinase-like activities. This is the first prokaryotic laccase sequenced and the first PPO showing such multipotent catalytic activity. The expression of several truncated products indicates that the four copper-binding sites typical of blue multicopper proteins are essential for the laccase activity of this enzyme. However, the last two of these sites are not necessary for tyrosine hydroxylase activity as this activity is retained in a truncated product containing the first two sites as well as the extra histidine-rich clusters close to the N-terminus of the protein.
Sanchez-Amat
A
A
Department of Genetics and Microbiology, University of Murcia, Spain.
Lucas-Elío
P
P
Fernández
E
E
García-Borrón
J C
JC
Solano
F
F
D000595:Amino Acid Sequence; D001419:Bacteria / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D004156:Catechol Oxidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005798:Genes, Bacterial; D042845:Laccase; D008522:Mediterranean Sea; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D010858:Pigmentation; D010957:Plasmids; D014871:Water Microbiology
D010088:Oxidoreductases; D004156:Catechol Oxidase; D042845:Laccase; D014442:Monophenol Monooxygenase