Compact Archaeal Variant of Heme a Synthase

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FEBS Letters 580 (2006) 5351–5356

Compact archaeal variant of heme A synthase

Anna Lewin*, Lars Hederstedt Department of Cell & Organism Biology, Lund University, So¨lvegatan 35, SE 22362 Lund, Sweden

Received 1 August 2006; revised 30 August 2006; accepted 30 August 2006

Available online 12 September 2006

Edited by Stuart Ferguson

number 2, 4, 6 and 8 each contains one invariant histidine res- Abstract The N- and C-terminal halves of the heme A synthase polypeptide of Bacillus subtilis, and many other organisms, are idue. Large extracytoplasmic loops, each containing two homologous. This indicates that these enzyme proteins originate invariant cysteine residues, connect transmembrane segments from a tandem duplication and fusion event of a gene encoding a 1 and 2, and segments 5 and 6. Based on the internal sequence protein half as large. The ape1694 gene of the hyperthermophilic similarity in CtaA it has been suggested that the ctaA gene, archaeon Aeropyrum pernix encodes a protein that is similar to encoding B. subtilis heme A synthase, is a result of a tandem the hypothetical small primordial protein. We demonstrate that gene duplication and fusion event [8]. this A. pernix protein is a heat-stable membrane bound heme B. subtilis CtaA protein isolated from B. subtilis strains over- A synthase designated cCtaA. The case of cCtaA is unusual in producing the protein or from recombinant E. coli cells, has evolution in that the primordial-like protein has not become ex- properties typical for cytochromes and contains heme B in tinct and apparently carries out the same function as the twice as substoichiometric amounts and various amounts of heme A large more diversified heme A synthase protein variant found in most cytochrome a-containing organisms. [8,9]. The bound heme A is thought to be the enzyme product Ó 2006 Federation of European Biochemical Societies. Published accumulating because it cannot be delivered to apo-cyto- by Elsevier B.V. All rights reserved. chrome a. It is unclear whether the heme B bound to CtaA functions as a prosthetic group or is an artifact generated by Keywords: CtaA; cCtaA; Heme A synthesis; ape1694; Protein the experimental systems. Enzyme substrate, heme O, and also evolution; Aeropyrum pernix the reaction intermediate heme I have been found in an isolated mutant variant of heme A synthase [5]. Aeropyrum pernix is a hyperthermophilic archaeon that was isolated in 1993 [10]. This bacterium has a strictly aerobic respiratory metabolism and two heme A-containing terminal 1. Introduction oxidases [11]. The open reading frame ape1694 in the A. pernix strain K1 genome sequence [12] encodes a predicted 161 resi- Heme A is exclusively found as a prosthetic group in termi- dues protein that is annotated as a ‘‘hypothetical cytochrome nal oxidases of aerobic respiratory chains, for example in cyto- aa3 controlling protein’’, but the sequence of this protein is chrome c oxidase in mitochondria and many bacteria [1,2]. similar to either half of B. subtilis CtaA (Fig. 2, panel A); Heme A is synthesized from heme B with heme O as a stable the protein has four predicted transmembrane segments and intermediate [3]. In the heme B to heme O synthesis step the a large extra membrane segment connecting helices number 1 vinyl side chain at C2 of heme B is modified into a hydroxy- and 2, and contains the histidine and cysteine residues that ethyl-farnesyl group. The heme O to heme A step is a modi- are invariant in bacterial CtaA orthologs. These features sug- fication of the methyl group at C8 of the porphyrin ring to a gested to us that the protein is a compact variant of the heme formyl group (Fig. 1). It seems as if heme O to heme A biosyn- A synthase polypeptide. We therefore, from here on, call this thesis occurs in three steps with two hydroxylated heme O protein cCtaA. intermediates (Fig. 1) [4,5]. First, the methyl group at C8 of To determine if the A. pernix cCtaA is a primordial-like var- heme O is oxygenated resulting in a monohydroxylated heme iant of heme A synthase polypeptide we have in this work O (heme I), which is then further oxidized to a dihydroxylated cloned the ape1694 gene and analyzed properties of cCtaA heme O. This dihydroxy intermediate is probably very unstable produced in E. coli. and the formyl group of heme A is obtained by the elimination of a water molecule. Heme A synthase is far from understood and defects in this enzyme in humans is associated with severe 2. Materials and methods multi-systemic infantile disorders [6,7]. Bacillus subtilis heme A synthase, CtaA, is a membrane pro- 2.1. Bacterial strains and plasmids Bacterial strains and plasmids used in this work are listed in Table 1. tein with 306 amino acid residues [8] (Fig. 2, panel A). It has The DNA techniques and the construction of plasmids pLAL5, eight predicted transmembrane a-helices [5] (Fig. 2, panel B, pLAL6 and pLAL7 are described in Appendix A. lower right). The N- and the C-terminal halves of the B. subtilis CtaA protein each has four transmembrane segments and 2.2. Media, bacterial growth and isolation of membranes show about 28% sequence identity. Transmembrane helices E. coli and B. subtilis strains were grown on LB agar (LA) or Tryp- tose blood agar base (Difco) plates. Liquid cultures were grown in LB or in Nutrient sporulation medium with phosphate [13] in agitated *Corresponding author. Fax: +46 2224113. (200 rpm) baffled Erlenmeyer-flasks. Cultures were grown at 37 °C. E-mail address: [email protected] (A. Lewin). Growth media were when needed supplemented with 50 lg/ml ampicil-

0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.08.080 5352 A. Lewin, L. Hederstedt / FEBS Letters 580 (2006) 5351–5356

Fig. 1. Structural formula of protoheme IX (heme B) and proposed heme O to heme A biosynthesis pathway. (A) Structure of heme B with the four pyrrole rings indicated by letters according to the Fisher nomenclature. (B) Proposed heme A synthase reaction steps.

B Primordial heme A Present day heme A synthase polypeptide synthases in procaryotes

Homodimer of small polypeptide heme A synthase, e.g. cCtaA

Large polypeptide Gene duplication heme A synthase, and fusion e.g.B. subtilis CtaA

Fig. 2. Comparison between A. pernix cCtaA and B. subtilis CtaA proteins. Panel A: The 306 residues CtaA sequence (Bs) aligned with the 161 residues cCtaA sequence (Ap) repeated in tandem. The N-terminal residue in the sequences is underlined. Residues that are invariant in CtaA of the closely related bacteria B. subtilis, B. cereus, B. anthracis, B. ﬁrmus, Staphylococcus aureus and S. epidermidis are indicated by stars above the B. subtilis sequence. Panel B: Predicted transmembrane topology and oligomeric state of A. pernix cCtaA and B. subtilis CtaA in the membrane, and a scheme illustrating the proposed evolution of heme A synthases from a primodial protein of unknown function. Conserved histidine and cysteine residues in A. pernix cCtaA and CtaA of Bacillus species are indicated in the topology models by dots and open squares, respectively.

lin or 50 lg/ml kanamycin or 12.5 lg/ml chloramphenicol for E. coli 20 mM Na-MOPS/HCl buffer, pH 7.4, containing 0.1% (w/v) Thesit and 4 lg/ml of chloramphenicol for B. subtilis. E. coli membranes were was used for gel-filtration. The flow rate was 30 ml per hour and isolated as described in Appendix A. 5 ml fractions were collected.

2.3. Puriﬁcation of A. pernix cCtaA and gel exclusion chromatography 2.4. Miscellaneous methods Isolation of A. pernix cCtaA from E. coli Top10/pLAL7 cells using Alignments were done using the Vector NTi, AlignX program. The Ni-NTA is described in Appendix A. A Sepharose S-300 column NCBI homepage (http://www.ncbi.nlm.nih.gov/) was used for BLAST (Pharmacia Biotech) (B = 2.7 cm, l = 105 cm) equilibrated with [14] searches. A. Lewin, L. Hederstedt / FEBS Letters 580 (2006) 5351–5356 5353

Table 1 Bacterial strains and plasmids used in this study Strain or plasmid Genotype properties Source of reference Strains Bacillus subtilis LMT20R trpC2, DctaA;SpR [5] Escherichia coli MM296 thi, pro, hsdR, supE4 [25] TOP10 mcrA, D(mrr-hsdRMS-mcrBC), recA, endA1, nupG;FÀ;SmR Invitrogen

Plasmids PCR-Blunt II-TOPO KmR Invitrogen pBAD-myc-His A AmR Invitrogen pHPKS pHP13 derivative; CmR,EmR [26] pCTA1305 pHP13 with ctaABC on a 4.9 kb fragment; CmR,EmR [9] pCTHI10 pHPKS derivative with ctaA including sequence encoding [5] a C-terminal hexa histidyl tag; CmR,EmR pLAL5 pHPKS with B. subtilis ctaA promoter region; CmR,EmR This work pLAL6 pLAL5 with ape1694 gene, including sequence encoding This work a C-terminal hexa histidyl tag cloned downstream of ctaA promoter; CmR,EmR pLAL7 ape1694 gene in pBAD-myc-His A; AmR This work SpR,SmR,KmR,AmR,CmR,EmR indicates resistance to spectinomycin, streptomycin, kanamycin, ampicillin, chloramphenicol and erythromycin, respectively.

SDS–PAGE and Immuno blot (Western blot) were done as described [15,16]. Blots were developed using SuperSignal west pico chemilumi- nescent substrate (Pierce). cCtaA peptide (corresponding to residues 45–60) rabbit antiserum was obtained from Eurogentec, Belgium. Both the peptide antiserum and anti-rabbit IgG peroxidase-linked sheep antiserum (Amersham Pharmacia) were used at a 2000-fold dilution. Isolation of CtaA from B. subtilis LMT20R/pCTHI10 was done as re- cently described [5]. Light absorption spectroscopy of membranes and isolated protein was done as described before [17]. Protein concentra- tions were determined using the bicinchoninic acid protein assay (Pierce Chemical Co.) with bovine serum albumin as the standard. Heme was extracted and analyzed by reversed-phase HPLC [5]. Pyridine hemochromogen analysis was preformed as described before [8]. N-terminal sequence analysis and quantitative amino acid hydrolysis were done at PAC, Karolinska Institutet, Stockholm.

3. Results

3.1. E. coli cells expressing ape1694 contain heme A To analyze whether the cCtaA protein functions as a heme A synthase the ape1694 gene of A. pernix strain K1 was cloned into the B. subtilis/E. coli shuttle vector pLAL5, resulting in pLAL6. The B. subtilis ctaA deletion strain LMT20R and E. coli strain MM294 were transformed with the two plasmids. Plasmid pLAL6 was not found to complement the heme A deficiency of strain LMT20R. The construction of pLAL6 and the details of the complementation experiment are presented in the Supplementary material. E. coli cells can synthesize heme O but not heme A [18]. Heme A was found in E. coli MM294/pLAL6, but not in MM294/pLAL5, as determined by HPLC analysis of hemes extracted from isolated membranes (Fig. 3). Strain MM294/ Fig. 3. HPLC elution profiles of hemes extracted from membranes of E. coli TOP10/pLAL7, MM294/pLAL6 and MM294/pLAL5. An A pLAL6 also contained monohydroxylated heme O (heme I). denotes heme A; B, heme B; O, heme O; and *, heme I. The identity of These findings indicated that cCtaA protein has heme A syn- the various types of hemes in the profile was confirmed by also running thase activity. Western blot analysis of the MM294/pLAL6 the extracts mixed with samples known to contain heme A, heme I or membranes, however, failed to detect cCtaA antigen suggest- heme O. The TOP10/pLAL7 membranes analyzed were isolated from cells grown over night in the presence of arabinose. ing that cCtaA protein was only produced in low amounts in this strain or as an unstable protein. To increase ape1694 gene expression in E. coli and have a LB and gene expression was induced by adding 0.02% (w/v) tightly regulated system, ape1694 was cloned into pBAD- arabinose to the medium. SDS–PAGE analysis of isolated myc-His A, resulting in pLAL7 which encodes a C-terminal membranes and staining for protein showed the presence of His-tagged cCtaA variant. E. coli TOP10 cells containing a 17 kDa polypeptide (a size expected for cCtaA) only in cells pLAL7 and pBAD-myc-His A, respectively, were grown in containing pLAL7 and grown in the presence of arabinose. 5354 A. Lewin, L. Hederstedt / FEBS Letters 580 (2006) 5351–5356

Western blot analysis of membrane polypeptides using cCtaA anti-peptide antiserum confirmed these result (Fig. S1). Membranes of E. coli TOP10/pLAL7 cells grown in the presence of arabinose had a red color compared to those of cells grown in the absence of arabinose and of TOP10/pBAD-myc- His cells. The difference (reduced minus oxidized) spectrum of TOP10/pLAL7 membranes showed a higher absorption inten- sity than that of TOP10/pBAD-myc-His membranes and the a-peak maximum was at 557 nm compared to 559 nm in the control (Fig. S2). These differences indicated that a cytochrome-like chromophore is associated with cCtaA. The heme B plus heme O contents of the TOP10/pBAD- myc-His A and TOP10/pLAL7 membranes were similar, 0.6 mol/g membrane protein, as determined by the pyridine hemochromogen. HPLC analysis of hemes extracted from membranes of the two strains showed the presence of large amounts of heme B and heme O, as expected. TOP10/pLAL7 membranes, however, also contained heme A and apparently Fig. 4. Difference (sodium dithionite reduced minus oxidized) light heme I (Fig. 3). The amounts of heme A and I in membranes absorption spectrum of isolated A. pernix cCtaA protein. The increased relative to the heme B content when cells were grown absorption scale is indicated by the bar. The Soret peak at 426 nm, in the presence of 0.02% arabinose over night as compared the b-peak of 527 nm and the a-peak at 557 nm, respectively, are to 4 h. indicated. The heme B plus heme O content of the analyzed sample was approximately 9 lM. The spectrum was recorded at room temperature using a 10 mm light path cuvette. 3.2. A. pernix cCtaA is a heme-binding protein The His-tagged cCtaA protein was solubilised from isolated TOP10/pLAL7 membranes using the non-ionic detergent The- fied enzymes were subjected to gel-permeation chromatography sit and purified by Ni-NTA chromatography as described in in the presence of 0.1% (w/v) Thesit. Both proteins eluted as a Appendix A. Preparations of isolated cCtaA protein contained monodisperse species and at a similar position in the chromato- one major protein with a mass of approximately 17 kDa gram with an apparent size of 380 k for the detergent micelle- (Fig. S3). N-terminal analysis of the protein yielded the protein complex (Fig. S5). From this result we conclude that sequence V/MKHLXI (the X indicates an unidentified amino the A. pernix and B. subtilis heme A synthases are of similar acid). This result compared to the expected MKHLRI N- size. Thus, the A. pernix protein forms a homo-oligomer and terminal sequence of cCtaA verified the identity of the isolated the assembled enzyme seemingly consists of twice as many protein. polypeptides as the B. subtilis heme A synthase. Preparations of isolated cCtaA contained 0.1 moles of heme B plus heme O per mole of cCtaA polypeptide, as determined by the pyridine hemochromogen combined with quantitative 4. Discussion amino acid analysis of acid-hydrolyzed sample. Purified cCtaA also contained small amounts of heme A and heme I (Fig. S4). In this work, we demonstrate that the A. pernix ape1694 gene The 412 nm/280 nm light absorption ratio of isolated cCtaA encodes heme A synthase. This finding is based on the following was 0.8. three observations. (i) The cCtaA sequence is very similar to The light absorption spectrum of cCtaA as isolated (fully that of either half of heme A synthase of B. subtilis, and many oxidized) showed a Soret peak at 412.5 nm. After reduction other bacteria, and residues known to be important for function with dithionite the spectrum showed maxima at 426 nm, are invariant. (ii) Heme A is found in recombinant E. coli cells in 527 nm and 557 nm. The redox difference (reduced minus oxi- which the cCtaA protein is present (E. coli cells normally cannot dized) spectrum of isolated cCtaA, shown in Fig. 4, is typical synthesize heme A). (iii) Isolated cCtaA protein binds heme O, for a low-spin cytochrome and similar to that of isolated heme I and heme A which are substrate, a reaction intermediate B. subtilis CtaA containing both heme B and O [5]. Reduced and product, respectively, for heme A synthase. heme A bound to B. subtilis CtaA shows an absorption peak A. pernix is a hyperthermophilic bacterium with optimal at 585 nm. Such a peak was not apparent in the cCtaA redox growth at 95 °C. The temperature dependency of cell-bound difference spectrum which is consistent with the low heme A enzymes for stability and activity generally reflects the optimal content of the sample. The light absorption spectrum of cCtaA temperature for growth of the organism. For example, after incubation at 80 °C for 20 minutes was found identical to A. pernix glutamate dehydrogenase has the activity maximum that of a not heat-treated sample. This shows that the protein at around 100 °C [19] and Aquifex aeolicus (optimal growth at was not denatured by the heat treatment and indicates extreme 95 °C) protoporphyrinogen oxidase shows little activity at thermo-stability of cCtaA. 30 °C and is most active in the temperature interval 60–90 °C [20]. We found that A. pernix heme A synthase is heat stable 3.3. cCtaA forms an oligomer and one can assume that the activity of this enzyme has a Purified B. subtilis CtaA in the detergent Thesit exists as temperature maximum of >60 °C. Low activity at 37 °C monomer or possibly dimer [8]. To compare the apparent can explain why cCtaA isolated from E. coli grown at this tem- molecular size of cCtaA (17 kDa polypeptide) and B. subtilis perature contained the substrate heme O and the reaction CtaA (34 kDa polypeptide) in detergent micelles the two puri- intermediate heme I. Similar to isolated B. subtilis CtaA, the A. Lewin, L. Hederstedt / FEBS Letters 580 (2006) 5351–5356 5355 purified cCtaA contained heme B. Another, or additional, pos- References sible reason for low activity of A. pernix heme A synthase in E. coli is that the available heme O has a farnesyl side chain [1] Michel, H., Behr, J., Harrenga, A. and Kannt, A. (1998) which is one isoprene unit shorter compared to heme O in Cytochrome c oxidase: structure and spectroscopy. Annu. Rev. Biophys. Biomol. Struct. 27, 329–356. A. pernix and other Archaea [21]. Heme A in A. pernix cyto- [2] Poole, R.K. and Cook, G.M. 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