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

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

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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: 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 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)bycopurification (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 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 Gram-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 O 2 and H 2 O [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- is V583 ⁄ 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 protoporphyrin 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 Ga 3+ and Fe 3+ 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 Cofactor-substituted catalase M. Brugna et al. 2664 FEBS Journal 277 (2010) 2663–2672 ª 2010 The Authors Journal compilation ª 2010 FEBS 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 His 6 -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 medium MIC (l M) Porphyrin concentration used in the growth medium (lM) a KatA polypeptide present b Co-PP > 150 8 + Cu-PP > 150 8 + Fe-PP > 150 8 + Ga-PP > 150 8 + Mg-PP > 150 2 + Ni-PP > 150 8 ) PP > 150 9 + Sn-PP > 130 7 + Zn-PP > 150 8 + Fe-meso < 15 8 + Pd-meso < 1.5 ND ND Ru-meso 4 1 + 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. M. Brugna et al. Cofactor-substituted catalase FEBS Journal 277 (2010) 2663–2672 ª 2010 The Authors Journal compilation ª 2010 FEBS 2665 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. faecalis or 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. faecalis V583 ⁄ 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-containing 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 present a Metal content (mol ⁄ mol KatA) PP content b (mol ⁄ mol KatA) Relative activity c (%) Co-PP-KatA ? Co, 0.70; Fe, 0.001 ND < 1 Cu-PP-KatA PP Cu, 0.04; Fe, < 0.01 1.1 2 Fe-PP-KatA Fe-PP Fe, 1.12 ND 100 Ga-PP-KatA Ga-PP Ga, 0.96; Fe, < 0.01 ND < 1 Mg-PP-KatA PP Mg, 0.02; Fe, 0.08 0.7 < 1 d PP-KatA PP Fe, 0.02 1.2 2 Sn-PP-KatA Sn-PP Sn, 0.76; Fe, 0.001 ND < 1 Zn-PP-KatA ? and PP Zn, 0.58; Fe, 0.02 0.075 3 Fe-meso-KatA Fe-meso Fe, 0.65 ND 35 Ru-meso-KatA ? Ru, 0.49; Fe, 0.05 ND < 1 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. c Specific enzyme activity with hydrogen peroxide as substrate relative to that of Fe-PP-KatA. d For some preparations, we found higher activity (up to 7%). Cofactor-substituted catalase M. Brugna et al. 2666 FEBS Journal 277 (2010) 2663–2672 ª 2010 The Authors Journal compilation ª 2010 FEBS 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 4 °C 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-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 4 °C 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 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-KatA, and Ru-meso-KatA) and the porphyrin-containing but metal-deprived (Cu-PP- KatA, Mg-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). M. Brugna et al. Cofactor-substituted catalase FEBS Journal 277 (2010) 2663–2672 ª 2010 The Authors Journal compilation ª 2010 FEBS 2667 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-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). Cofactor-substituted catalase M. Brugna et al. 2668 FEBS Journal 277 (2010) 2663–2672 ª 2010 The Authors Journal compilation ª 2010 FEBS 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 Fe 3+ 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. M. Brugna et al. Cofactor-substituted catalase FEBS Journal 277 (2010) 2663–2672 ª 2010 The Authors Journal compilation ª 2010 FEBS 2669 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. faecalis V583 cells containing plasmid pLUF15 (a deriv- ative of plasmid pAM401 containing the katA–his 6 gene 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 )1 NaCl, 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 His 6 -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. Bacteria were grown at 37 °C under oxic conditions. 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 centrifugation, at 5000 g for 30 min, when the cultures 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 37 °C with shaking for 1 h, and then broken in a French pressure cell operated at 16 000 p.s.i. The His 6 -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 manufac- turer’s instructions. The cytoplasmic fraction, 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 His 6 -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 His 6 -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 )1 chloramphenicol 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 37 °C 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 mixture of 2.1 m pyridine, 0.075 m NaOH and 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 Cofactor-substituted catalase M. Brugna et al. 2670 FEBS Journal 277 (2010) 2663–2672 ª 2010 The Authors Journal compilation ª 2010 FEBS 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. References 1 Milgrom GL (1997) The Colours of Life: an Introduction to the Chemistry of Porphyrins and Related Compounds. Oxford University Press, Oxford. 2 Frau´ stio JJR & Williams RJ (1991) The Biological Chemistry of the Elements. Clarendon Press, Oxford. 3 Kumar S & Bandyopadhyay U (2005) Free haem toxic- ity and its detoxification systems in human. Toxicol Lett 157, 175–188. 4 Hamza I (2006) Intracellular trafficking of porphyrins. ACS Chem Biol 1, 627–629. 5 Stojiljkovic I, Evavold BD & Kumar V (2001) Antimi- crobial properties of porphyrins. 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Infect Immun 70, 4494–4500. 31 Sone N & Fujiwara Y (1991) Effects of aeration during growth of Bacillus stearothermophilus on proton pump- ing activity and change on terminal oxidases. J Biochem (Tokyo) 110, 1016–1021. 32 Falk JE (1964) Porphyrins and Metalloporphyrins. Elsevier, Amsterdam. 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. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Cofactor-substituted catalase M. Brugna et al. 2672 FEBS Journal 277 (2010) 2663–2672 ª 2010 The Authors Journal compilation ª 2010 FEBS . 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. thus excluding many metal-substi- tuted porphyrins. For the in vivo production of catalase containing haem analogues, we grew E. faecalis strain V583 ⁄

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