Molecular cloning and functional characterization of a unique multipotent polyphenol oxidase from Marinomonas mediterranea

Marinomonas mediterranea is a recently isolated melanogenic marine bacteriu m containing laccase and tyrosinase activities. These activities are due to the expression of two polyphenol oxidases (PPOs), a blue multicopper lacca se and an SDS-activated tyrosinase. The gene encoding the first one, herein denominated M. mediterranea PpoA, has been isolated by transposon mutagene sis. cloned and expressed in Escherichia roll. Its predicted amino acid seq uence shows the existence of a signal peptide and four copper-binding sites characteristic of the blue multicopper proteins, including all fungal lacc ases. In addition, two additional putative copper-binding sites near its N- terminus are also present. Recombinant expression in E. coli of this protei n clearly demonstrates its multipotent capability, showing both laccase-lik e and tyrosinase-like activities. This is the first prokaryotic laccase seq uenced and the first PPO showing such muitipotent catalytic activity. The e xpression of several truncated products indicates that the four copper-bind ing sites typical of blue multicopper proteins are essential for the laccas e activity of this enzyme. However. the last two of these sites are nor nec essary for tyrosine hydroxylase activity as this activity is retained in a truncated product containing the first two sites as well as the extra histi dine-rich clusters close to the N-terminus of the protein. (C) 2001 Elsevie r Science B.V. All rights reserved.