Tài liệu Báo cáo khoa học: Phenol hydroxylase from Acinetobacter radioresistens S13 Isolation and

Phenol hydroxylase from Acinetobacter radioresistens S13

Isolation and characterization of the regulatory component

Ersilia Griva1

, Enrica Pessione1

, Sara Divari1

, Francesca Valetti1

, Maria Cavaletto2

, Gian Luigi Rossi3

and Carlo Giunta1

1

Dipartimento di Biologia Animale e dell’Uomo, Universita` di Torino, Italy; 2

Dipartimento di Scienze e Tecnologie Avanzate,

Universita` del Piemonte Orientale, Alessandria, Italy; 3

Dipartimento di Biochimica e Biologia Molecolare,

Universita` di Parma, Italy

This paper reports the isolation and characterization of

the regulatory moiety of the multicomponent enzyme

phenol hydroxylase from Acinetobacter radioresistens S13

grown on phenol as the only carbon and energy source.

The whole enzyme comprises an oxygenase moiety

(PHO), a reductase moiety (PHR) and a regulatory

moiety (PHI). PHR contains one FAD and one iron￾sulfur cluster, whose function is electron transfer from

NADH to the dinuclear iron centre of the oxygenase. PHI

is required for catalysis of the conversion of phenol to

catechol in vitro, but is not required for PHR activity

towards alternative electron acceptors such as cyto￾chrome c and Nitro Blue Tetrazolium. The molecular

mass of PHI was determined to be 10 kDa by SDS/

PAGE, 8.8 kDa by MALDI-TOF spectrometry and

18 kDa by gel-permeation. This finding suggests that the

protein in its native state is a homodimer. The isoelectric

point is 4.1. PHI does not contain any redox cofactor

and does not bind ANS, a fluorescent probe for hydro￾phobic sites. The N-terminal sequence is similar to those

of the regulatory proteins of phenol hydroxylase from

A. calcoaceticus and Pseudomonas CF 600.

In the reconstituted system, optimal reaction rate was

achieved when the stoichiometry of the components was 2

PHR monomers: 1 PHI dimer: 1 PHO(abc) dimer. PHI

interacts specifically with PHR, promoting the enhancement

of FAD fluorescence emission. This signal is diagnostic of a

conformational change of PHR that might result in a better

alignment with respect to PHO.

Keywords: regulatory proteins; multicomponent mono￾oxygenase; phenol hydroxylase.

Acinetobacter radioresistens S13 is able to grow on phenol

as the sole carbon and energy source via the ortho-pathway

(b-ketoadipate pathway). The first enzyme involved in

phenol degradation is phenol hydroxylase (PH), a mono￾oxygenase utilizing NADH as electron donor.

In previous studies we have found that the enzyme is

composed of three moieties which are readily separated by

chromatographic steps: the oxygenase (PHO), composed of

two heterotrimers (abc) (S. Divari, F. Valetti, P. Caposio, E.

Pessione, M. Calvaletto, E. Griva, G. Gribaudo, G. Gilardi

& C. Giunta, unpublished observation), the reductase

(PHR) [1] and a third protein (PHI) that is described in

this work.

A similar molecular composition has been found in

phenol hydroxylases from Pseudomonas CF 600 [2] and

A. calcoaceticus NCIB 8250 [3] and in toluene-2-mono￾oxygenase from Burkholderia cepacia [4], as well as in the

soluble methane monooxygenases (MMOs) from Methylo￾coccus capsulatus [5], Methylosinus trichosporium [6], Meth￾ylocystis sp.M [7] and in alkene monooxygenase from

Nocardia corallina [8].

In phenol hydroxylase of A. radioresistens S13, the third

component is needed for the overall enzyme activity; in

phenol hydroxylase from Pseudomonas CF 600, it promotes

substrate–oxygenase interaction [9]; in MMOs it alters the

local environment and the redox potential of the catalytic

centre [6,10–12].

Interestingly, in other aromatic monooxygenases (i.e.

toluene-4-monooxygenase from Pseudomonas mendocina

[13], toluene/o-xylene monooxygenase from Pseudomonas

stutzeri [14] and alkene monooxygenase from Xantobacter

Py2 [15]), two proteins, rather than one, are present besides

the oxygenase and the reductase moieties. In this case one has

a regulatory function, the other is a Rieske-type ferredoxin.

The question was asked whether, in A. radioresistens

S13, PHI promotes the overall catalytic activity of the

Correspondence to C. Giunta, Via Accademia Albertina,

13, 10123 Torino, Italy. Fax: + 39 0116704692,

E-mail: [email protected]

Abbreviations: ANS, 8-anilinonaphtalene-1-sulfonic acid ammonium

salt; CV, circular voltammetry; DPV, differential pulse voltammetry;

MCD, magnetic circular dicroism; MMO, methane monooxygenase;

MMOB, methane monooxygenase regulatory component; MMOH,

methane monooxygenase hydroxylase; MMOR, methane mono￾oxygenase reductase component; NBT, nitro blue tetrazolium;

PH, phenol hydroxylase; PHI, phenol hydroxylase regulatory protein;

PHR, phenol hydroxylase reductase; PHO, phenol hydroxylase

oxygenase; T2M, toluene-2-monooxygenase.

Enzymes: Phenol hydroxylase (EC 1.14.13.7); benzoate dioxygenase

(EC 1.14.12.10); toluene 4-monooxygenase (EC 1.14.14.1); toluene

2-monooxygenase (EC 1.14.13.-); alkene monooxygenase

(EC 1.14.13.-); xylene monooxygenase (EC 1.14.14.1); phthalate

dioxygenase (EC 1.14.12.7); p-hydroxybenzoate hydroxylase

(EC 1.14.13.2); toluene dioxygenase (EC 1.14.12.11); methane

monooxygenase (EC 1.14.13.25).

(Received 18 December 2002, accepted 6 February 2003)

Eur. J. Biochem. 270, 1434–1440 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03505.x