Báo cáo khoa học: In vivo production of catalase containing haem analogues pot

In vivo production of catalase containing haem analogues

Myriam Brugna*, Lena Tasse and Lars Hederstedt

Microbiology Group, Department of Biology, Lund University, Sweden

Introduction

Haem is present in most organisms as the prosthetic

group of proteins such as cytochromes, oxygenases,

haemoglobins, and catalases [1]. The versatile chemical

reactivity of the iron ion is expanded in the haem

molecule, giving the wide functional range of haem

proteins [2].

The physicochemical properties of the haem mole￾cule are, however, troublesome for cells [3]. At neutral

pH, haem is an amphiphilic, poorly water-soluble

molecule. In the reduced state, in the presence of

molecular oxygen, haem is also a potent catalyst for

reactive oxygen species formation. These toxic effects

must be avoided when haem is transported and stored

within cells. Haem, synthesized within the cell or

acquired from the environment, and destined to finally

be a prosthetic group of a haem protein, is therefore

Keywords

antibacterial agents; catalase;

Enterococcus faecalis; haem protein;

metal porphyrins

Correspondence

L. Hederstedt, Department of Biology,

Biology Building, Lund University,

So¨lvegatan 35, SE-22362 Lund, Sweden

Fax: +46 46 222 41 13

Tel: +46 46 222 86 22

E-mail: [email protected]

Present address

*Laboratoire de Bioe´nerge´tique et Inge´nierie

des Prote´ines, Institut de la Me´diterrane´e,

CNRS, Marseille cedex 20, France; Univer￾site´ de Provence, 3 Place Victor Hugo,

Marseille cedex 3, France

Laboratoire des Interactions Plantes-Micro￾organismes, UMR INRA-CNRS, Chemin de

Borde-Rouge BP 52627, Castanet-Tolosan,

France

(Received 4 March 2010, revised 31 March

2010, accepted 9 April 2010)

doi:10.1111/j.1742-4658.2010.07677.x

Haem (protohaem IX) analogues are toxic compounds and have been con￾sidered for use as antibacterial agents, but the primary mechanism behind

their toxicity has not been demonstrated. Using the haem protein catalase

in the Gram-positive bacterium Enterococcus faecalis as an experimental

system, we show that a variety of haem analogues can be taken up by bac￾terial cells and incorporated into haem-dependent enzymes. The resulting

cofactor-substituted proteins are dysfunctional, generally resulting in

arrested cell growth or death. This largely explains the cell toxicity of haem

analogues. In contrast to many other organisms, E. faecalis does not

depend on haem for growth, and therefore resists the toxicity of many

haem analogues. We have exploited this feature to establish a bacterial

in vivo system for the production of cofactor-substituted haem protein vari￾ants. As a pilot study, we produced, isolated and analysed novel catalase

variants in which the iron atom of the haem prosthetic group is replaced

by other metals, i.e. cobalt, gallium, tin, and zinc, and also variants con￾taining meso-protoheme IX, ruthenium meso-protoporphyrin IX and

(metal-free) protoporphyrin IX. Engineered haem proteins of this type are

of potential use within basic research and the biotechnical industry.

Structured digital abstract

l MINT-7722358, MINT-7722368: katA (uniprotkb:Q834P5) and katA (uniprotkb:Q834P5)

physically interact (MI:0915) by copurification (MI:0025)

Abbreviations

Co-PP, cobalt protoporphyrin IX; Cu-PP, copper protoporphyrin IX; Fe-meso, iron meso-protoporphyrin; Fe-PP, protohaem IX; Ga-PP, gallium

protoporphyrin IX; Mg-PP, magnesium protoporphyrin IX; MIC, minimal inhibitory concentration; Ni-PP, nickel protoporphyrin IX; Pd-meso,

palladium meso-protoporphyrin IX; PP, protoporphyrin IX; Ru-meso, ruthenium meso-protoporphyrin IX; Sn-PP, tin protoporphyrin IX;

Zn-PP, zinc protoporphyrin IX.

FEBS Journal 277 (2010) 2663–2672 ª 2010 The Authors Journal compilation ª 2010 FEBS 2663