title: A new multicopper oxidase from Gram-positive bacterium Rhodococcus erythropolis with activity modulating methionine rich tail.
abstract: Multicopper oxidases are involved in a wide variety of physiological tasks in nature. They are part of the lignin formation/decomposition system in plants and fungi. In bacteria they are part of developmental processes and the heavy metal resistance apparatus. A well characterised example is the copper tolerance protein CueO of Escherichia coli (CueO(EC)). Here, we report the heterologous expression of the apo- and holo-form of CueO(RE), a homologue to CueO(EC) from Rhodococcus erythropolis. Upon incubation with copper(II) ions, low active apo-CueO(RE) was converted into the active holo-CueO(RE) in vivo. The holo-form was physico-chemically characterised using a copper(I) BCA complex and the model substrate 2,6-dimethoxyphenol. The spectroscopic and catalytic properties are different from CueO(EC), revealing a high catalytic efficiency (k(cat)/K(m)) of 115 min(-1)mM(-1) with physiological K(m) of 80 μM for the cuprous oxidase activity. At the C-terminus of CueO(RE) a methionine rich tail region was identified which can be found in a variety of actinobacteria. Chimeras of the E. coli and R. erythropolis enzymes were constructed to investigate the influence of this tail regarding kinetic parameters. It was shown that the tail did not have the same function as the corresponding methionine rich loop in CueO(EC). However, it modulated the kinetic properties of the enzyme.
authors.affiliation: Insitut für Bioorganische Chemie, Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich, D-52426 Jülich, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Classen
authors.forename: Jörg
authors.initials: J
authors.lastname: Pietruszka
authors.forename: Saskia Marina
authors.initials: SM
authors.lastname: Schuback
mesh_terms: D000595:Amino Acid Sequence; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D007700:Kinetics; D008715:Methionine / Q000737:chemistry*; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification*; D017434:Protein Structure, Tertiary; D012240:Rhodococcus / Q000201:enzymology*
chemical_list: D003300:Copper; D008715:Methionine; D010088:Oxidoreductases