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

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;

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: Lars.Hederstedt@cob.lu.se

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 vivosystem 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.

most likely transiently bound to specific protein factors

in cells Little is known about intracellular haem

trans-port and how haem is incorporated into proteins

in vivo to form haem proteins in various subcellular

locations [4]

Haem analogues, with a metal ion other than iron

or with structural alterations in the porphyrin

macro-cycle, are generally toxic to cells, and have been

con-sidered for use as antibacterial agents [5] The

molecular basis for the toxicity of haem analogues and

the resistance of some bacteria against such

com-pounds have not been fully explained In this work, we

have investigated the potential of the Gram-positive

bacterium Enterococcus faecalis to import different

haem analogues from the growth medium and

incorpo-rate these into catalase apo-protein in the cytoplasm to

form substituted catalase This research was performed

to find the explanation for the general toxicity of haem

analogues, so that we can better judge these types of

compound as potential antimicrobial and antitumour

drugs A second purpose was to increase our

know-ledge of the mechanisms of the uptake and

intracellu-lar transport of haem A third aim was to establish an

in vivo system for the production of structurally

complex haem proteins containing synthetic

metallo-porphyrins

Six features of E faecalis make this bacterium very

suitable for our investigations: (a) the bacterium can

take up haem [6] and a variety of haem analogues

from the growth medium (this work); (b) as E faecalis

is a positive bacterium, there is, unlike in

Gram-negative bacteria such as Escherichia coli, no outer

membrane constituting a possible barrier for the

uptake of porphyrin compounds; (c) E faecalis does

not synthesize haem; (d) E faecalis does not depend

on haem for growth, and grows well also without

haem; (e) if supplied with haem, the bacterium is

capable of aerobic respiration [7–10] and produces

two haem proteins – a membrane-bound cytochrome

bd respiratory quinol oxidase [7], and a cytoplasmic

monofunctional catalase [6]; and (f) the growth of

many bacteria is inhibited by noniron

metalloporphy-rins, but E faecalis strains are generally resistant to

this type of compound ([11] and this work)

Catalase (EC 1.11.1.6) is present in most aerobic

organisms, and serves in part to protect the cells from

the toxic effects of hydrogen peroxide by catalysing its

decomposition into O2 and H2O [12,13] E faecalis

catalase is a typical monofunctional catalase [14] It is

a homotetrameric enzyme containing one

proto-haem IX (Fe-PP) molecule per KatA polypeptide of

478 amino acids The structure of the E faecalis

enzyme is rather complex (Fig S1) Each monomer

has an extended arm-like N-terminal domain and a major globular C-terminal domain, with the haem-con-taining active site deeply buried in a b-barrel structure The four subunits are tightly associated with the N-ter-minal domain of one monomer woven into the globu-lar domain of the neighbouring subunit Attempts to accomplish the assembly of catalase in vitro from its constituents, (polypeptide and Fe-PP) have so far failed [15]

Results

Susceptibility of E faecalis to haem analogues The recombinant E faecalis strain V583⁄ pLUF15 used

in this work carries, on a plasmid, a variant of the

E faecalis katA gene, resulting in overproduction of hexahistidyl-tagged KatA polypeptide [6] The tag allows catalase to be purified from disrupted cells in a single affinity chromatographic step

Production of catalase in E faecalis requires haem

in the growth medium In a previous study, we found that the maximal amount of catalase in E faecalis V583⁄ pLUF15 is obtained in the presence of about

10 lm haemin [6] Stojiljkovic et al [11] reported that growth of E faecalis cells is resistant to 17 different tested noniron porphyrins, but minimal inhibitory con-centrations (MICs) for the different compounds were not provided To determine the relative toxicities of haem analogues and find the concentrations at which

E faecalis would grow well, we cultivated E

faecal-isV583⁄ pLUF15 in TSBG (a haem-free medium) sup-plemented with different concentrations of the compounds of interest The MIC value obtained for the metal-substituted haem analogues cobalt proto-porphyrin IX (Co-PP), copper protoporphyrin IX (Cu-PP), Fe-PP, gallium protoporphyrin IX (Ga-PP), magnesium protoporphyrin IX (Mg-PP), nickel proto-porphyrin IX (Ni-PP), and zinc protoproto-porphyrin IX (Zn-PP), and for protoporphyrin IX (PP), was

> 150 lm, and for tin protoporphyrin IX (Sn-PP)

it was > 130 lm, i.e., these compounds are not toxic to E faecalis Ruthenium meso-protoporphyrin

IX (Ru-meso) and iron meso-protoporphyrin IX (Fe-meso) were found to be somewhat toxic, and palla-dium meso-protoporphyrin IX (Pd-meso) very toxic (Table 1)

In vivo synthesis of gallium-substituted catalase

Ga3 +and Fe3 +are similar in size and form a very stable complex with PP In contrast to iron, the gallium ion is not oxidized or reduced under physiological

conditions Ga-PP is very toxic to Gram-negative

bacteria (MIC < 2 lm) and some Gram-positive

bacte-ria such as Staphylococcus species (MIC < 3 lm) [11]

Ga-PP seems to be transported into Gram-negative

bacteria via haem uptake systems [11] The toxicity of

Ga-PP has been suggested to result from the

incorpora-tion of this haem analogue into haem proteins, which

thereby become nonfunctional, but this has not been

experimentally demonstrated [5] Reasons for the

resistance of E faecalis to Ga-PP (Table 1) could be

that this haem analogue is not taken up by the

bacte-rium, or that Ga-PP is incorporated into haem proteins

but has no drastic effect because E faecalis cells are

not dependent on haem proteins for growth

To investigate whether E faecalis cells can take up

haem analogues from the growth medium and

incorpo-rate them into protein in the cytoplasm, we first tested

whether catalase substituted with Ga-PP can be

pro-duced E faecalis V583⁄ pLUF15 was grown in the

presence of 8 lm Ga-PP, and in parallel in the absence

of any porphyrin compound and in the presence of

8 lm Fe-PP, respectively, as controls The cytoplasmic

fraction was isolated from the cells and analysed for

KatA polypeptide by immunoblot (Fig 1) We have

previously shown that KatA protein is only found in

cytoplasmic extracts of E faecalis if the prosthetic

group has been incorporated [6] In its absence, the

KatA polypeptide is probably not completely folded

and is therefore degraded The cytoplasmic fraction

from E faecalis V583⁄ pLUF15 grown in the absence

of a porphyrin compound completely lacked KatA antigen, as expected (Fig 1, lane B) Extracts from cells grown in the presence of Fe-PP (Fig 1, lane C)

or Ga-PP (lane I) contained KatA These results sug-gested that Ga-PP is incorporated into the KatA poly-peptide to form a gallium-substituted catalase protein (Ga-PP-KatA)

Purification and characterization of Ga-PP-KatA The His6-tagged iron-containing (Fe-PP-KatA) and Ga-PP-KatA catalases were purified from cell-free extracts by using metal affinity chromatography The purity of preparations was evaluated by SDS⁄ PAGE, which, for both Fe-PP-KatA and Ga-PP-KatA, showed one polypeptide band corresponding to KatA with an apparent molecular mass of 54 kDa (Fig 2)

In the case of Ga-PP-KatA, an additional protein band of about 110 kDa was observed This band cor-responds to KatA dimer, as determined by immuno-blot analysis (Fig 1, lane I)

Amino acid analysis of preparations of isolated Fe-PP-KatA and Ga-PP-KatA confirmed the composi-tion of KatA polypeptide as deduced from the katA

Table 1 Toxicity of porphyrins, porphyrin concentrations used for

growth and presence of catalase protein in E faecalis V583⁄

pLUF15 ND, not done.

Porphyrin added

to the growth

Porphyrin concentration used in the growth medium (l M ) a

KatA polypeptide present b

a Concentration used in the growth medium for production of KatA.

b As determined by immunoblot with cell extracts (see Fig 1).

Fig 1 KatA immunoblot of cytoplasmic fraction from E

faecal-is V583 ⁄ pLUF15 grown in TSBG medium (lane B) or TSBG medium supplemented with the indicated porphyrin compounds (lanes C–M) The concentrations of porphyrin used in the growth medium are provided in Table 1 Lanes A and N each contained

80 ng of purified haem-containing E faecalis catalase (Fe-PP-KatA) Lanes B–M each contained 2 lg of total cytoplasmic protein.

Fig 2 SDS ⁄ PAGE of preparations of isolated E faecalis normal and gallium-substituted catalase Lane A: molecular mass markers (kDa) Lane B: 2 lg of Fe-PP-KatA Lane C: 1 lg of Ga-PP-KatA The gel was stained for protein with Coomassie brilliant blue.

sequence, and was used for quantitative determination

of KatA protein Metal analysis combined with the

protein analysis showed the presence of 1.12 mol of

iron atoms per mol of KatA polypeptide in

Fe-PP-KatA Isolated Ga-PP-KatA contained 0.96 mol of

gallium per mol of KatA polypeptide, and only trace

amounts of iron (Table 2)

Porphyrin present in Fe-PP-KatA and Ga-PP-KatA

was extracted from the isolated catalase proteins by

using acid⁄ acetone, and analysed by RP-HPLC The

porphyrin of Ga-PP-KatA eluted as a single peak at

7.0 min Reference Ga-PP had the same retention time

Fe-PP and the porphyrin extracted from Fe-PP-KatA

both eluted at 8.5 min This showed that Fe-PP and

Ga-PP are present in the isolated proteins, and

excluded the possibility that these metalloporphyrins

added to the culture are modified during transport into

E faecalisor after being incorporated into KatA

poly-peptide, as is the case for catalase HPII of E coli [16]

Enzymatic and spectroscopic properties of

Ga-PP-KatA

Isolated Ga-PP-KatA showed less than 1% catalase

activity as compared with Fe-PP-KatA (Table 2)

Enzyme activity measurement with the cytoplasmic cell

fraction from E faecalisV583⁄ pLUF15 containing

Ga-PP-KatA as compared with that containing

Fe-PP-KatA showed the same relative results (data not

shown) This confirmed that Ga-PP-KatA is essentially

inactive, and that this is not due to inactivation during

isolation of the protein

Light absorption spectra of the purified

iron-con-taining and gallium-coniron-con-taining catalases are presented

in Fig 3A,B Fe-PP-KatA showed a Soret peak at

406 nm and weak absorption bands at 504, 541 and

625 nm These features are characteristic for haem-containing catalases [17] Ga-PP-KatA showed a very different spectrum, with the Soret peak at 422 nm and distinct absorption maxima at 548 and 588 nm

Screening for in vivo production of cofactor-substituted catalase The results obtained with Ga-PP and the properties of Ga-PP-KatA demonstrated that E faecalis cells can take up a haem analogue from the growth medium and incorporate it into KatA polypeptide to form cofactor-substituted catalase To determine whether this is a general property, we grew E faecalis V583⁄ pLUF15 cells in the presence of Co-PP, Cu-PP, Mg-PP, Ni-PP, Sn-PP, and Zn-PP The bacteria were also grown in the presence of Fe-meso, Ru-meso, and

PP The concentrations of porphyrins used in the growth medium are given in Table 1 In the case of Ru-meso, a low concentration had to be used because

of the toxicity of this compound

Production of catalase was determined by immuno-blot analysis of cytoplasmic fractions (Fig 1; Table 1) KatA protein was obtained with all of the porphyrins tested, except for Ni-PP, indicating that various haem analogues can be inserted into the protein

The catalase proteins were isolated by the same procedure as used for Fe-PP-KatA and Ga-PP-KatA The resulting preparations were pure or contai-ned some contaminating proteins (in the cases of Co-PP-KatA, Sn-PP-KatA, and Fe-meso-KatA) as evaluated by SDS⁄ PAGE (gel not shown) The cata-lase activities of purified proteins are presented in Table 2 Fe-meso-KatA showed 35% activity as Table 2 Properties of isolated normal and cofactor-substituted catalases ND, not done.

Variant

Porphyrin presenta

Metal content (mol ⁄ mol KatA)

PP content b

(mol ⁄ mol KatA)

Relative activityc(%)

a Porphyrin found in isolated catalase protein as determined by HPLC, light absorption spectroscopy, and fluorometry A question mark indi-cates that the identity of the porphyrin(s) has not been established b Protoporphyrin content determined by fluorescence measurements.

ND, not done.cSpecific enzyme activity with hydrogen peroxide as substrate relative to that of Fe-PP-KatA.dFor some preparations, we found higher activity (up to 7%).

compared with Fe-PP-KatA The other proteins lacked

detectable activity or showed very low activity

Covalently bound KatA dimers (in addition to

monomeric KatA) were found in preparations of

iso-lated cofactor-substituted catalases (Fig 2) and also in

those of isolated Fe-PP-KatA that had been stored at

4C for several weeks In most cases, the dimers did

not constitute more than 20% of the total KatA, but

for Ga-PP-KatA and Sn-PP-KatA, they could

repre-sent a major form of the protein Dimers were not

observed in the case of Co-PP-KatA and

Fe-meso-KatA (Fig 1, lanes E and G) PP-Fe-meso-KatA also formed

dimers, indicating that their formation is not

porphyrin metal-dependent It has been reported that

lyophilization or storage of catalase in solution enhances dimer formation [18] Intermolecular disulfide crosslinks are formed in porcine erythrocyte catalase when the enzyme is stored at 4C for more than

1 week [19] Disulfide bond formation can be excluded

in the case of E faecalis catalase, because this protein does not contain any cysteine, and proteins were reduced before SDS⁄ PAGE

All isolated catalase variants were analysed for cobalt, copper, iron, gallium, magnesium, ruthenium, tin and zinc content The major metal found in each preparation was generally the same as that contained

in the metalloporphyrin added to the growth medium (Table 2) Notable exceptions, however, were the prep-arations of isolated Cu-PP-KatA and Mg-PP-KatA, which contained only low amounts of metals; Cu-PP-KatA contained 0.04 mol of copper per mol of Cu-PP-KatA polypeptide, and Mg-PP-KatA contained 0.02 mol of magnesium per mol of KatA Similarly, the prepara-tion of isolated PP-KatA contained little metal,

< 0.08 mol of metal per mol of KatA polypeptide, except for zinc, which was found at 0.13 mol per mol

of KatA In the following, we deal separately with the metal-substituted (Co-PP-KatA, Sn-PP-KatA, Zn-PP-KatA, Fe-meso-Zn-PP-KatA, and Ru-meso-KatA) and the porphyrin-containing but metal-deprived (Cu-PP-KatA, Mg-PP-(Cu-PP-KatA, and PP-KatA) catalases

Characterization of metal-substituted catalases Porphyrins present in the different preparations with metal-substituted catalase were analysed by RP-HPLC For Fe-meso-KatA and Sn-PP-KatA, the chromato-gram of the extracted porphyrin completely agreed with that of the reference compound, i.e Fe-meso (retention time of 5.5 min) and Sn-PP (retention time

of 13 min), respectively In the cases of Co-PP-KatA, Zn-PP-KatA, and Ru-meso-KatA, the chromatograms

of the extracted porphyrins showed complex patterns that did not entirely correspond to those of the refer-ence compounds (data not shown) Some metallopor-phyrins, e.g Zn-PP, lose the metal ion under acidic conditions, but this was not the reason for the com-plexity observed in the chromatograms

Light absorption spectra of purified Co-PP-KatA, Sn-PP-KatA, Zn-PP-KatA and Ru-meso-KatA were all different and distinct from that of Fe-PP-KatA (Fig 3A,B) (maxima at 430, 544 and 577 nm for Co-PP-KatA, at 420, 551 and 590 nm for Sn-PP-KatA,

at 421, 554, 574, 629 and 670 nm for Zn-PP-KatA, and at 404, 526, 559 and 677 nm for Ru-meso-KatA) The spectrum of Fe-meso-KatA was similar to that of Fe-PP-KatA, but with slightly shifted absorption

Fig 3 Light absorption spectra of isolated normal and

cofactor-substituted catalases Porphyrins added to the growth medium to

produce the various catalases are indicated (A) The Soret band

region (380–470 nm) (B) The region from 500 to 700 nm (C)

Spectra of catalases produced in the presence of Cu-PP, Mg-PP,

and PP The intensities of these spectra have been normalized

with respect to the Soret band absorption The inset in (C)

shows a magnified view of the spectra between 460 and

720 nm The absorption scale in each panel is indicated by a

vertical bar The proteins were in 50 m M potassium phosphate

buffer (pH 8.0).

maxima (396, 499, 531 and 619 nm as compared with

406, 504, 541 and 625 nm)

The fluorescence emission spectrum of the porphyrin

contained in Zn-PP-KatA, in a pyridine⁄ water ⁄

NaOH⁄ Tween-80 mixture, was not similar to that of

the reference solution of Zn-PP in the same solvent,

indicating that the bound compound is not Zn-PP

(Fig 4) Moreover, fluorescence spectral analysis

showed that isolated Zn-PP-KatA contained a small

amount of PP (0.075 mol of PP per mol of KatA

poly-peptide) (Fig 4; Table 2) Zn-PP-KatA contained

0.58 mol of zinc per mol of KatA polypeptide

(Table 2) These results, taken together, indicate that

zinc in Zn-PP-KatA is bound to a nonfluorescent,

unidentified porphyrin compound This conclusion is

consistent with the light absorption spectrum of

Zn-PP-KatA, which presents features reminiscent of

the spectrum of PP-KatA (maxima at 554, 574 and

629 nm) (see next section) and features of the

unidenti-fied zinc porphyrin (maxima at 421 and 670 nm)

(Fig 3A–C)

Characterization of catalase obtained with PP in

the growth medium

The HPLC chromatogram of porphyrin extracted from

PP-KatA showed one major species, with the same

retention time (20 min) as PP (data not shown) To

further characterize and analyse the amount of

porphyrin present in PP-KatA, the isolated protein was diluted into a pyridine⁄ water ⁄ NaOH ⁄ Tween-80 mixture to denature the protein and dissolve the por-phyrin The fluorescence emission spectrum of the solution was identical to that of PP in the same solvent (maximum at 632 nm) (Fig 4) This confirmed the HPLC data showing that PP-KatA contained PP On the basis of fluorescence measurements and compari-son with standard solutions of PP, a stoichiometry of approximately 1 mol of PP per mol of KatA polypep-tide was found (Table 2) These results demonstrated that PP (metal-free porphyrin) is taken up by the bac-terial cell and incorporated into catalase protein The light absorption spectrum of the isolated PP-KatA showed absorption maxima at 416, 517, 554, 574 and 628 nm (Fig 3C) This spectrum has features very similar to those described for Proteus mirabilis catalase produced in E coli, which contains a mixture of Fe-PP and PP [20] The X-ray crystal structure of this cata-lase shows that PP can replace haem, and that this has essentially no effect on the architecture of the active site PP has also been found bound to the chlorophyll biosynthetic protein BchH of Rhodobacter capsulatus expressed in E coli [21]

Catalase obtained with Cu-PP and Mg-PP in the growth medium

Unexpectedly, catalases produced during growth of

E faecalis V583⁄ pLUF15 in the presence of Cu-PP or Mg-PP (Fig 1, lanes D and M) contained only trace amounts of copper and magnesium (Table 2) Light absorbance spectroscopy (Fig 3C) and fluorescence spectroscopy (Fig 4) showed that PP was the major porphyrin in the two proteins Approximately 1 mol of

PP and 0.7 mol of PP per mol of KatA polypeptide were found in Cu-PP-KatA and Mg-PP-KatA, respec-tively (Table 2)

These findings suggested that the metal ion of the porphyrin is removed during transport of the porphy-rin from the medium to the catalase protein in the cytoplasm Alternatively, but less likely, the metal-loporphyrin is incorporated into catalase and the metal

is subsequently lost from the protein, leaving the PP bound to the protein

Discussion

Our findings demonstrate, first, that the haem pros-thetic group of catalase can be replaced by various haem analogues Moreover, they explain the general cellular toxicity of noniron metalloporphyrins They also show the potential of the bacterium E faecalis as

Fig 4 Fluorescence emission spectra of PP, Mg-PP, Cu-PP, and

Zn-PP, and of the porphyrins contained in isolated PP-KatA,

Mg-PP-KatA, Cu-PP-Mg-PP-KatA, and Zn-PP-KatA The excitation wavelengths

used are indicated in Experimental procedures Emission spectra

peak maxima are indicated by dotted lines The solvent was

pyri-dine ⁄ water ⁄ NaOH ⁄ Tween-80 (see Experimental procedures for

details).

an in vivo system for the production of

cofactor-substi-tuted haem proteins

It is sometimes desirable to replace the metal of a

metalloporphyrin in a specific protein For example,

investigations on the reaction mechanisms of

haem-containing enzymes and electron transfer in proteins

are greatly aided if the metal of the normal prosthetic

group can be substituted The photosensitivity of, for

example, zinc, tin and magnesium porphyrins provides

a convenient way of initiating a reaction very quickly

[22] Other reasons to modify the prosthetic group are

to search for proteins with novel properties suitable

for various biotechnical applications, e.g the design of

sensors, and for structural analysis by, for example,

NMR, where Fe3+ can strongly interfere with the

analysis through being paramagnetic [23]

The haem group of some water-soluble proteins can,

in vitro, be removed and reinserted, or substituted for

another metalloporphyrin, such as Zn-PP or Co-PP

[24] The assembly of more complex haem proteins,

such as membrane-bound respiratory enzymes or

cata-lase, however, can, at present, generally not be

accom-plished in vitro The lack of knowledge concerning the

biogenesis of haem proteins makes it difficult to design

experimental conditions under which haem in a

com-plex haem protein can be substituted in vitro Various

approaches have been used to construct artificial haem

enzymes [25] Woodward et al [26] recently presented

a method for the incorporation of haem analogues into

protein using a haem-permeable E coli strain that is

unable to biosynthesize haem The usefulness of this

method is, however, restricted to compounds that are

not toxic to E coli, thus excluding many

metal-substi-tuted porphyrins

For the in vivo production of catalase containing

haem analogues, we grew E faecalis strain V583⁄

-pLUF15, which overproduces catalase polypeptide, in

the presence of the respective haem analogue in the

growth medium Noniron metalloporphyrin

com-pounds are generally toxic to bacteria [11], but, with

the notable exception of Pd-meso, they do not inhibit

growth of E faecalis (Table 1) This allowed us to

pro-duce and isolate catalases in which the normal iron

atom is replaced by other metals, i.e cobalt, gallium,

tin, and zinc We could also produce catalase proteins

containing Fe-meso and Ru-meso Supplementation of

the haem-free growth medium with PP resulted, much

to our surprise, in PP-containing catalase, i.e protein

containing a metal-free porphyrin group

The hemH gene (Ef1989) of E faecalis V583

appar-ently encodes a ferrochelatase Ferrochelatases catalyse

the last step in haem synthesis, i.e the insertion of

ferrous iron into PP As growth of bacteria in the

presence of PP did not result in catalase containing Fe-PP, it appears that the hemH gene is not expressed,

or that the HemH protein lacks ferrochelatase activity with PP Lactococcus lactis and E faecalis are closely related bacteria L lactis contains a hemH gene (called hemZ), and can apparently take up Fe-PP from the growth medium Indirect evidence with a HemZ-defi-cient strain suggests that HemZ is a ferrochelatase [27] Thus, an alternative explanation for our results obtained with E faecalis is that the hemH gene does encode a functional and expressed ferrochelatase, but that iron is not available in sufficient amounts in the cell to allow Fe-PP synthesis Supplementation of the TSBG growth medium with both PP and iron chloride, however, did not result in Fe-PP-KatA being found in

E faecalis

The molecular machinery responsible for staphylo-coccal haem acquisition is encoded by the genes of two distinct membrane-associated transport systems, the iron-regulated surface determinant system, and the haem transport system [28] Iron-regulated surface determinant system-like proteins are present in Bacillus anthracis and Listeria monocytogenes, suggesting that they function in haem uptake in these Gram-positive pathogens [29] The Gram-positive bacteria Streptococ-cus pyogenes and Corynebacterium diphtheriae contain the HmuTUV proteins for haem acquisition [30] Hmu-TUV proteins are similar to the proteins involved in haem transport in Gram-negative bacteria, and are pro-posed to be components of an ATP-binding cassette-type transporter E faecalis V583 contains HmuTUV,

as indicated by the genome sequence, and possibly, in parallel with other, as yet unknown, carriers, it might transport both haem and haem analogues If so, these transport systems are rather promiscuous, much like the E faecalis catalase protein, in binding porphyrins

It is not known how haem is transported intracellularly

in bacteria, and the mechanisms by which haem is incorporated into soluble and membrane-bound pro-teins inside cells have not been determined [4] It is evi-dent from our results that these putative transport components also work with haem analogues The toxic-ity of noniron metallo-PPs for bacteria has been sug-gested, but not previously demonstrated, to result from these compounds being taken up into the cell and incorporated into vital haem proteins instead of haem [5] We show here that haem analogues are indeed taken up and can be incorporated into haem proteins, resulting in their inactivation Pd-meso and, to a lesser extent, Ru-meso were found to be toxic also for

E faecalis (Table 1) This toxicity might be connected

to meso-protoporphyrin IX, as the bacterium tolerated Fe-PP better than Fe-meso

The semisynthetic catalases that we have produced

in the present investigation are metalloproteins of a

type hitherto not described Our results show that the

E faecalis cell can be used for the in vivo production

of novel haem proteins Following the same general

approach, one may be able to generate a great variety

of complex soluble and membrane-bound substituted

haem proteins

Experimental procedures

Bacterial strains and growth conditions

E faecalisV583 cells containing plasmid pLUF15 (a

under control of the native promoter) [6] was grown in

TSBG [15 gÆL)1 tryptone, 5 gÆL)1 soytone peptone (both

from Lab M, Bury, UK), 5 gÆL)1NaCl, 1% (w⁄ v) glucose,

30 mm Mops (pH 7.4), and 5 mm potassium phosphate

buffer, pH 7.0] TSBG contains less than 0.05 lM Fe-PP

[6] Chloramphenicol was added to a final concentration of

20 mg⁄ L For the production of normal and

metal-substi-tuted His6-tagged catalases, the TSBG medium was

supple-mented with various porphyrins Co-PP, Cu-PP, Ga-PP,

Mg-PP, Ni-PP, Sn-PP, Zn-PP, Fe-meso and Ru-meso were

purchased from Porphyrin Products (Logan, UT, USA)

and were considered to be pure Haemin and PP were

obtained from Sigma Chem Co Pd-meso was a kind

gift from S Vinogradov (University of Pennsylvania,

Philadelphia) Porphyrins were dissolved in

dimethylsulfox-ide in the cases of Co-PP, Cu-PP, Ga-PP, Ni-PP, Sn-PP,

Zn-PP, Fe-meso, and Ru-meso, in Tween-80 [1.5% (w⁄ v) in

water] in the cases of Mg-PP and PP, or in Tween-20

[12.5% (v⁄ v) in an alkaline solution] in the case of Fe-PP

Preparative cultures were grown in 1 L portions in 5 L

baf-fled Erlenmeyer flasks at 200 r.p.m in a rotary incubator

Cultures with porphyrins added were protected from light

to avoid possible photoeffects The cells were harvested by

reached late exponential growth phase as determined from

the attenuance measured at 600 nm (D600 nm)

Isolation of cytoplasmic cell fraction and

purification of catalase

The cytoplasmic fraction of E faecalis cells was prepared

essentially as described previously [6], except in the case of

large-scale preparations The cells from 3 L cultures were

suspended in 50 mm potassium phosphate buffer (pH 8.0),

incubated with 1 mgÆmL)1 lysozyme at 37C with shaking

for 1 h, and then broken in a French pressure cell operated

at 16 000 p.s.i The His6-tagged catalases were isolated from

the cytoplasmic cell fraction, corresponding to a 3 L culture,

with a 1 mL HiTrap chelating column (Pharmacia Bio-tech), loaded with nickel ions, according to the

supplemented with 300 mm NaCl and 1 mm histidine, was loaded onto the affinity column, equilibrated in 50 mm potassium phosphate buffer (pH 8.0) containing 300 mm NaCl and 1 mm histidine This buffer was used to wash the column, and His6-tagged catalase was eluted from the matrix by raising the histidine content of the buffer to

50 mm The purified catalases were dialysed against 50 mm potassium phosphate buffer (pH 8.0) and stored on ice

We have previously shown that the His6-tag at the C-ter-minal end of KatA does not interfere with the function of the protein [6]

MIC determinations

Bacteria were grown in TSBG medium supplemented with

20 mgÆL)1chloramphenicol and different concentrations of the porphyrin to be tested The media were inoculated to a D600 nm of 0.15 with a fresh culture grown in unsupple-mented TSBG The cultures (3 mL) were incubated at

37C for 8 h, under oxic conditions, in the dark The MIC was defined as the lowest concentration of porphyrin that prevented growth All experiments were carried out in trip-licate The amount of dimethylsulfoxide, Tween-80 or Tween-20 added to the medium as solvent for the porphy-rin compound did not affect bacterial growth

Fluorescence spectroscopy

Purified substituted catalase proteins were denatured in a

0.0075% (w⁄ v) Tween-80 in water After centrifugation at

10 000 g for 10 min to remove the denatured proteins, the fluorescence emission spectra between 530 and 750 nm of the solutions were recorded on a Shimadzu RF 5301 PC spectrofluorophotometer, using excitation wavelengths of

406 nm for PP and Cu-PP, 424 nm for Zn-PP, and 418 nm for Mg-PP The excitation and emission slits were 3 and

10 nm, respectively Known concentrations of PP dissolved

in the same solvent as the samples were used for calibration

of the fluorometer readings

Other methods

Extraction of porphyrins from purified catalase protein and analysis by RP-HPLC were performed as described by Sone and Fujiwara [31] Protein concentrations were determined using the bicinchoninic acid assay (Pierce Chem Co.), with BSA as the standard Concentrations of KatA were determined by the combined use of quantitative amino acid analysis (performed at the Department of Laboratory Medi-cine, Malmo¨ University Hospital), pyridine haemochromogen

analysis [32], and rocket immunoelectrophoresis

Immuno-blot and rocket immunoelectrophoresis were performed

with rabbit anti-KatA serum as previously described [6]

Catalase activity was assayed as described previously [6]

Light absorption spectra were recorded with a

Shima-dzu UV-2101PC spectrophotometer Metal content analysis

was performed by using inductively coupled plasma MS

Acknowledgements

This work was supported by grant 621-2007-6094 from

the Swedish Research Council and a grant from the

Crafoord Foundation M Brugna was the recipient of

a EU Marie Curie long-term fellowship (contract

HPMF-CT-2000-00918) We are grateful to S

Vinog-radov (University of Pennsylvania, Philadelphia, PA)

for the generous gift of Pd-meso and to T Olsson

(Lund University, Sweden) for the metal content

anal-ysis We thank I Sta˚l for expert technical assistance

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Supporting information

The following supplementary material is available: Fig S1 Crystal structure of E faecalis catalase This supplementary material can be found in the online version of this article

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