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Biochimica et Biophysica Acta 1797 (2010) 1933–1939

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Biochimica et Biophysica Acta

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Ubiquinol-binding site in the : Mutagenesis reveals features important for substrate binding and inhibition

Mary S. Albury, Catherine Elliott, Anthony L. Moore ⁎

Department of Chemistry & Biochemistry, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK

article info abstract

Article history: The alternative oxidase (AOX) is a non-protonmotive that is found in all plants, some Received 6 November 2009 fungi, green algae, bacteria and pathogenic protozoa. The lack of AOX in the mammalian host renders this Received in revised form 11 January 2010 an important potential therapeutic target in the treatment of pathogenic protozoan infections. Accepted 12 January 2010 Bioinformatic searches revealed that, within a putative ubiquinol-binding crevice in AOX, Gln242, Asn247, Available online 18 January 2010 Tyr253, Ser256, His261 and Arg262 were highly conserved. To confirm that these amino-acid residues are important for ubiquinol-binding and hence activity substitution mutations were generated and char- Keywords: Alternative oxidase acterised. Assessment of AOX activity in isolated Schizosaccharomyces pombe mitochondria revealed that Ubiquinone-binding mutation of either Gln242, Ser256, His261 and Arg262 resulted in >90% inhibition of antimycin A-insensitive Structure–function relations respiration suggesting that hydroxyl, guanidino, imidazole groups, polar and charged residues in addition to Site-directed mutagenesis the size of the amino-acid chain are important for ubiquinone-binding. Substitution of Asn247 with Schizosaccharomyces pombe mitochondria glutamine or Tyr253 with phenylalanine had little effect upon the respiratory rate indicating that these residues are not critical for AOX activity. However replacement of Tyr253 by alanine resulted in a 72% loss of activity suggesting that the benzoquinone group and not hydroxyl group is important for quinol binding. These results provide important new insights into the ubiquinol- of the alternative oxidase, the identity of which maybe important for future rational drug design. © 2010 Elsevier B.V. All rights reserved.

1. Introduction now considered to be important potential therapeutic targets in these systems [9]. In addition to its ubiquitous presence in all plants [1,2], the It is generally accepted that AOX is a non-haem di-iron carboxylate cyanide-insensitive terminal alternative oxidase (AOX) is also found protein in which the metal atoms are ligated by glutamate and in species of fungi, green algae, bacteria and protozoa and more histidine residues within a 4-helix bundle [1,10,11]. The current recently, in molluscs, nematodes and chordates but not in mammals model of the AOX predicts a monotopic integral membrane protein [3,4]. Of particular importance, however, is its presence in pathogenic [1,10,11] associating with one leaflet of the bilayer. Moreover protozoa such as the blood parasite Trypanosoma brucei [5] the this model is supported by extensive site-directed mutagenesis intestinal parasites Cryptosporidium parvum [6,7] and Blastocystis studies [11–17] and EPR spectroscopy has confirmed the presence hominis and the emerging recognition that the AOX also plays a of a binuclear iron centre [18,19]. More recently an FTIR study [20] of critical role in the life cycle of yeasts such as Candida albicans [8]. the redox centres of a highly purified and stable preparation of Although the exact function of AOX within these organisms is recombinant AOX (rTAO) from Trypanosoma brucei [21] confirmed uncertain it is generally considered that the oxidase either plays a the presence of a di-iron centre. This study revealed clear spectro- key role in the reoxidation of cytosolic NADH (trypanosomes) [5] or in scopic signatures of two distinct redox processes associated with the the oxygen defence mechanism in the case of the anaerobic parasites full reduction of oxygen through to water. Spectroscopic signatures or yeasts [4]. It should be noted that to date there are no specific contained features that could be assigned to the protonation of at least inhibitors or treatments of trypanosomiasis or cryptosporidiosis and one carboxylate group, further perturbations of carboxylic and current treatments of the former often have fatal side effects [5,7]. histidine residues, which to our knowledge, is the first direct evidence Because of their absence in the mammalian host, AOX are to confirm that the alternative oxidase is indeed a di-iron carboxylate protein [20]. In addition to an for the reduction of oxygen to water, the AOX must contain a binding site for ubiquinol, the reducing Abbreviations: AOX, alternative oxidase; TAO, trypanosomal alternative oxidase; Q(H ), 2 substrate of the protein. The structures of several quinone-binding (reduced) ubiquinone; SHAM, salicylhydroxamic acid – ⁎ Corresponding author. Tel.: +44 1273 678479; fax: +44 1273 678433. proteins have been determined to atomic resolution [22 24] E-mail address: [email protected] (A.L. Moore). however, the general architecture of a quinone-binding site remains

0005-2728/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2010.01.013 1934 M.S. Albury et al. / Biochimica et Biophysica Acta 1797 (2010) 1933–1939 elusive. Despite this, several characteristics thought to be important underlined. Each full length mutant AOX was excised on a BspHI– for quinone-binding have been identified. Elucidation of the quinone- BamHI fragment and ligated to the yeast expression vector pREP1/N binding site in the membrane associated cytochrome bc1 complex has (a modified version of pREP1 [29,31] in which the NdeI site was resulted in a model comparable to quinone-binding sites in replaced with NcoI) which had been digested with NcoI and BamHI, photosynthetic reaction centres in which the quinone-binding pocket yielding pREP1-Q242N, pREP1-N247Q, pREP1-Y253A, pREP1-S256T, is formed by the ends of two transmembrane helices thus placing the pREP1-H261A and pREP1-R262K. ubiquinone ring near the membrane interface [24,25]. Aromatic residues identified close to quinone-binding regions may interact 2.3. General molecular biology procedures with the quinone head group in a parallel stacking manner. Additionally, Abramson et al. [24] observed a novel quinone-binding Oligonucleotides were obtained from MWG Biotech. Mutations domain containing a group of polar residues exposed to the interior of were originally identified by restriction analysis and confirmed by the membrane that are uniquely conserved amongst ubiquinol sequencing (Beckman Coulter Genomics). Full length clones were also oxidases. A sequence analysis of respiratory and photosynthetic sequenced to confirm the absence of nonspecific mutations. S. pombe complexes that react with quinones has resulted in the identification cells were transformed using a modified lithium acetate procedure of a putative quinone-binding motif, consisting of an HR (His-Arg) based on the method of Okazaki et al. [32]. Other procedures were as pair and a triad element [26]. In addition to residues highlighted by described by Sambrook et al. [33]. Fisher and Rich [26], serine, arginine and tyrosine residues have been identified by biochemical means as being critical for quinone-binding 2.4. Cell growth and mitochondrial isolation in SQR (succinate:ubiquinone ) [27–29]. We [1] and others [10,11] proposed a model for the ubiquinol- Transformed S. pombe cells were batch-cultured in glucose binding site in AOX based on accumulated data and bioinformatics minimal medium [34] supplemented with 0.4 mM adenine and searches, which identified a hydrophobic pocket, between α-helices II 0.7 mM uracil. Cells were grown in the absence of thiamine to and III, which could act as a channel enabling the substrate to gain promote AOX expression under the control of the nmt promoter [31]. access to the active site from the membrane-binding domain. Mitochondria were isolated using the method outlined in [35] with In this paper we investigate the validity of this model through a the modifications described in [36]. Mitochondrial protein concentra- series of site-directed mutagenesis experiments of residues identified tions were determined using bicinchoninic acid with BSA as a as being important for ubiquinol-binding through a bioinformatics standard [37]. search. The study has revealed that Gln242, Ser256, His261 and Arg262 are critical for ubiquinol-binding and Asn247 maybe impor- 2.5. Oxygen uptake tant for inhibition. Respiratory activity was measured with a Clark-type electrode (Rank Brothers, Cambridge, U.K.) using 0.1 to 0.5 mg mitochondria 2. Materials and methods suspended in 0.4 mL air-saturated reaction medium (250 µM at 25 °C [38]) containing 0.65 M mannitol, 20 mM MOPS/NaOH (pH 6.8), 2.1. Strains 1 mM MgCl2, 5 mM Na2HPO4 and 10 mM NaCl. The S. pombe strain used was Sp.011 (ade6–704, leu1–32, ura4- − 2.6. Western analysis D18, h ). The E. coli strains DH5α and JM110 were used for amplification of plasmids. Separation of mitochondrial proteins on non-reducing SDS- polyacrylamide gels, transfer to nitrocellulose membranes and 2.2. Site-directed mutagenesis and plasmid construction detection of AOX protein using monoclonal antibodies raised against the S. guttatum AOX was performed as described previously [30]. Construction of pREP1-AOX and pREP1-Y253F, (used to express wild-type AOX and the Y253F mutant in S. pombe) have been 3. Results described previously [13,30]. Mutagenesis of AOX was performed using the Quick Change mutagenesis kit (Stratagene) according to On the basis that the alternative oxidase is a monotopic protein manufacturer's instructions, with plasmid pSLM-AOR [13]. Table 1 [10,11] which associates with one leaflet of the lipid bilayer in a shows oligonucleotides used for each mutation, with altered codons manner similar to that proposed for prostaglandin H2 synthase and squalene synthase, two monotopic proteins for which the crystal structure has been solved [39,40], we undertook a bioinformatic Table 1 approach to identify potential regions of AOX sequence which are Oligonucleotides used for site-directed mutagenesis to produce mutated forms of S. guttatum AOX. likely to associate with the membrane. Alignments of >100 AOX sequences available to date have been compared with (1) other Mutation Primer sequence members of the membrane-bound di-iron carboxylate proteins, Q242N 5′-CTGGTGCTGGCGGTGAACGGGGTCTTCTTCAAC-3′ comprising the plastid terminal oxidases (PTOX) [41] and the CLK1 5′-GTTGAAGAAGACCCCGTTCACCGCCAGCACCAG-3′ homologue, Coq7 [42] and (2) the soluble di-iron carboxylate N247Q 5′-CAGGGGGTCTTCTTCCAGGCCTACTTCCTGG-3′ proteins, including Δ9desaturase, the R2 subunit of ribonucleotide 5′-CCAGGAAGTAGGCCTGGAAGAAGACCCCCTG-3′ Y253A 5′-CCTACTTCCTGGGGGCCCTGCTCTCCCCC-3′ reductase (R2) and the hydroxylase component of methane mono- 5′-GGGGGAGAGCAGGGCCCCCAGGAAGTAGG-3′ (MMOH). We searched for homology/insertions/deletions S256T 5′-CTGGGGTACCTGCTCACCCCCAAGTTCGCCCAC-3′ in AOX sequences relative to these other proteins and took into ′ ′ 5 -GTGGGCGAACTTGGGGGTGAGCAGGTACCCCAG-3 account hydropathy, proposed secondary structural elements and the H261A 5′-CCCCCAAGTTCGCCGCCCGGGTTGTGGGC -3′ 5′-GCCCACAACCCGGGCGGCGAACTTGGGGG-3′ distribution of polar aromatic (Trp, Tyr) and basic residues (Lys, Arg) R262K 5′-CCAAGTTCGCCCACAAGGTTGTGGGCTACC-3′ which are prevalent at the interfacial regions of membrane proteins 5′-GGTAGCCCACAACCTTGTGGGCGAACTTGG-3′ (e.g. [43]). We also analysed membrane-binding regions of monotopic Oligonucleotides used for site-directed mutagenesis with the altered codon in bold and proteins assigned from crystal structures of prostaglandin H2 underlined. Note that the Y253F mutated AOX has been described previously (see Ref 13). synthase, squalene synthase and fatty acid acyl [39,40,44]. M.S. Albury et al. / Biochimica et Biophysica Acta 1797 (2010) 1933–1939 1935

Table 2 others, based on the criteria outlined above and on that basis the most Regions of AOX sequence identified by a bioinformatic approach as potential candidates hydrophobic region of the protein is region A. Analysis of the for membrane-binding regions. sequence reveals it is flanked by Trp/Tyr and Arg/Lys residues and Region Residues Sequence and key features may form an aromatic-sided helix. It appears to be more hydrophobic A 231–255 [RWYERALVLAVQGVFFNAYFLGYLL]. The most hydrophobic in AOX/PTOX/Coq7 sequences than in soluble di-iron carboxylate region of the protein, flanked by Trp/Tyr and Arg/Lys. May form proteins and we would therefore propose it to be the major an aromatic-sided helix. More hydrophobic in AOX/PTOX/Coq7 membrane-binding region of AOX. than in soluble di-iron carboxylate proteins. Proposed to be the Based on modelling and bioinformatics searches, we and others major membrane-binding region of AOX. B 180–190 [VAAVPGMVGGVLLH] The C-terminus of the first helix in the [1,10,11,45,46] have previously proposed that the sequence upstream putative four-helix bundle. Hydrophobic face, a proline residue from region A (Table 2), which lies between α-helices II and III (see may disrupt this helix. More hydrophobic in AOX/PTOX/Coq7 Fig. 1), forms a hydrophobic pocket within the protein which acts as a than in the soluble di-iron carboxylate proteins. Possibly channel enabling its substrate, ubiquinol, to gain access to the active comprises a secondary membrane-binding region of AOX, site from the membrane-binding domain. Furthermore the structural perhaps by indirect binding via intra-molecular association. C 292–303 [PAPAIALDYWRL] Short region, this predicted helix is flanked model shown in Fig. 1 is consistent with the known non-proto- by Trp/Tyr/Arg residues. Represents an insertion relative to the nmotive nature of ubiquinol oxidation in the alternative oxidase [47] soluble di-iron carboxylates and is absent in Coq7. Predicted to and the hydrophobic pocket is in close proximity to the membrane be interfacial based on hydropathy analysis. Rich in Pro surface and the active site of the protein. The 21 amino-acids within residues. Not included in the current AOX model [16]. D 100–130 [VSYWAVPPSKEDGSEWRWTCFRPWETYQA] Region not this region contain residues likely to form H-bonds with carbonyl included in structural model [16]. Ill-defined predicted groups of ubiquinone such as His and Ser in addition to those that secondary structure, but rich in aromatic and Pro residues. Not have the ability to stabilise ubiquinol-binding through π–π interac- predicted to be hydrophobic, perhaps associates with interface tions with benzoquinone ring such as Phe/Tyr/Arg, the majority of only. May form dimerising region. which have been identified by biochemical means as being critical Numbering and sequence information refer to S. guttatum AOX — see Fig. 1 for for quinone-binding in SQR (succinate:ubiquinone oxidoreductase) – locations. Regions have been ranked from A D in order of anticipated importance. [27–29]. The region also contains residues identified through muta- Based on a hydropathy analysis, A and B, have previously been identified as potentially interacting with the membrane [see refs. 10 and 11]. genesis studies as being important for inhibitor sensitivity in the alternative oxidase [48]. Specifically the region (Fig. 1) contains an HR pair (His261/ Based on this analysis, four regions of AOX that we consider likely to Arg262), previously identified by Fisher and Rich [26] as part of a be important for interactions of AOX with the inner mitochondrial potential hydroquinone-binding motif, and two of the three residues membrane were identified. These sequences are outlined in Table 2 identified by inhibitor-resistant screening as being important in and depicted in Fig. 1 and include the two regions of AOX that, due to ubiquinol-binding [48], flank the HR pair. Tyr253, a highly conserved their hydropathy, have previously been proposed to interact with the residue that we have previously postulated to be important in membrane [10,11]. Each region of sequence has been scrutinised and ubiquinol-binding [1,45], is positioned in the hydrophobic pocket ranked in terms of its potential for membrane binding, relative to the proximal to the HR pair. Importantly this region also contains Gln242 and Asn247, the only significantly polar residues in this hydrophobic region, which we have previously shown to be different to that seen in the trypanosomal AOX isozymes [1,46], variation of which appears to result in differences in sensitivity to the inhibitor ascofuranone [16]. The putative quinone-binding domain also contains polar and charged residues similar to those of the ubiquinol oxidase of E. coli [24] and also a highly conserved serine residue (Ser256) which appears to play a key role in quinone-binding of complex II [27,28]. Fig. 2 shows a multiple sequence alignment of the putative ubiquinol-binding site from a variety of species including both thermogenic and non-thermogenic plants, metazoa, , proteobacteria and parasites. What is very striking from this multiple alignment is the extent to which Gln242, Tyr253, His261 and Arg262 are fully conserved across all species. Other residues that show significant conservation include, Ser256, Pro257 and Arg/Lys258. Given the high degree of conservation, residues Gln242, Tyr253, His261 and Arg262 in addition to Asp247 and Ser256 were chosen for mutagenesis. In this study, rather than mutating all of these residues to alanine more subtle changes were introduced in order to probe the relative importance of the hydroxyl groups (in the case of Tyr253 and Ser256), the guanidino group (Arg262) and, in order to maintain the polar nature, conservative changes in the case of Gln242 and Asp247. His261, however, was mutated to alanine since the methyl group of this residue is not capable of forming hydrogen bonds with the carbonyl group of ubiquinone. Table 3 summarises the results obtained when this series of site- Fig. 1. Diagrammatic representation of the proposed secondary structure arrangement directed mutations of the Sauromatum guttatum alternative oxidase of the alternative oxidase. This figure represents membrane topology and location of were expressed in S. pombe. The extent to which the mutation affected the hydrophobic crevice within the C-terminal portion of the AOX. Spheres represent alternative oxidase activity in isolated S. pombe mitochondria was iron atoms. Note that the first 164 residues of the mature protein along with residues assessed by measuring the rate of oxygen uptake in the presence of 286–303 are not modeled as they do not have equivalents in Δ9-desaturase. A–D refer to the hydrophobic regions identified in Table 2 whereas the amino-acids in red are antimycin A and ascertaining the sensitivity to the alternative oxidase those proposed to be important for ubiquinol-binding. inhibitor, octylgallate. Since respiratory activity is not necessarily 1936 M.S. Albury et al. / Biochimica et Biophysica Acta 1797 (2010) 1933–1939

Fig. 2. Alignment of the proposed Q binding region of AOX sequences from a number of different species, grouped by phyla. Conserved residues are shown in colour — R/K in red, H in blue, Q/N in green, Y in teal and P in yellow. Note, Q binding region is defined as residues 236–264 as per Sauromatum guttatum numbering (P22185). Alignment created using ClustalW and viewed using SeaView. Full species names and accession (UniProt) numbers — Arum concinnatum B9X258; Nelumbo nucifera C0SUJ4; Philodendron bipinnatifidum Q65YQ8; Sauromatum guttatum P22185; Symplocarpus renifolius Q5KSN9; Dracunculus vulgaris Q65YS0; Arabidopsis thaliana O23913; Daucus carota B1P5D1; Glycine max O82518; Oryza sativa subsp. japonica Q8W855; Saccharum officinarum Q66PX0; Triticum aestivum Q8S913; Zea mays Q8GT27; Aspergillus niger O74180; Candida albicans O93853; Coprinopsis cinerea A8NDS4; Chaetomium globosum Q2GZL4; Coccidioides immitis Q1E8R2; Cryptococcus neoformans var. grubii Q8NKE2; Gelasinospora sp. (strain S23) Q8J1Z2; Neurospora crassa Q01355; Penicillium chrysogenum Q6TBA7; Uncinocarpus reesii C4JFI2; Trichoplax adhaerens B3RQ52; Anadara ovalis C5HYP9; Nematostella vectensis A7RXL2; Acanthamoeba castellanii Q07CY9; Aliivibrio salmonicida (strain LFI1238) B6EGE6; Erythrobacter sp. NAP1 A3WI95; Methylophaga thiooxidans C0N2S5; Novosphingobium aromaticivorans (strain DSM 12444) Q2G807; Octadecabacter antarcticus 238 B5K463; Photobacterium profundum 3TCK Q1YY39; Phenylobacterium zucineum (strain HLK1) B4R9G7; Roseobacter denitrificans Q164N3; Roseovarius sp. TM1035 A6DV40; Sphingomonas wittichii (strain RW1/DSM 6014/JCM 10273) A5VGI7; Thiobacillus denitrificans Q3SHK3; Thioalkalivibrio sp. K90mix B9ZS20; Vibrio campbellii AND4 A8TAF3; Vibrio harveyi A7MXL7; Vibrio shilonii AK1 A6D0V8; Blastocystis hominis (not in database yet); Cryptosporidium hominis Q5CGN5; Cryptosporidium parvum Q761U6; Trypanosoma brucei brucei Q26710; Perkinsus marinus C5K4Z3.

Table 3 Effect of site-directed mutagenesis on alternative oxidase activity in S. pombe controlled exclusively by AOX activity, but also by that of substrate mitochondria. dehydrogenases [49], we used a cocktail of substrates (NADH, succinate, and glutamate in the presence of ATP) as we have shown Oxidase Activity nmolO2/min/mg % Control previously that in the presence of these substrates Q-reducing capacity Wt 54±8 100 is maximal and AOX exerts more than 95% of total flux control over Q242N 3±1 6 N247Q 52±6 96 antimycin A-resistant NADH oxidation in isolated S. pombe mitochon- Y253F 33±6 61 dria [49]. It should be noted that, in the absence of antimycin A, Y253A 15±3 28 respiratory control ratios greater than 2, following addition of FCCP, S256T 4±2 7 were observed with all of the mutants indicating that the mutations H261A 3±1 5 fi R262K 3±1 6 speci cally affected the alternative pathway and not the main cytochrome pathway or mitochondrial integrity. What is immediately Antimycin A-insensitive oxygen-uptake rates in S. pombe mitochondria containing obvious from Table 3 is that Gln242, Ser256, His261 and Arg262 are either wild-type or mutated AOX proteins. Rates (±standard error) were measured in the presence of 2 µM antimycin A, 2 mM NADH, 9 mM succinate, 0.2 mM ATP and 9 mM structurally critical for alternative oxidase activity since even glutamate. Percentages refer to the respective rates relative to AOXwt. conservative substitution results in greater than 93% inhibition of M.S. Albury et al. / Biochimica et Biophysica Acta 1797 (2010) 1933–1939 1937 activity. In the case of Gln242, which is fully conserved across all species possessing the alternative oxidase (Fig. 2), replacement with asparagine results in an inactive protein even though the polar nature of the substitution is maintained. Unlike the Q242N mutation, replacement of N247 with glutamine, however, resulted in an active protein. Such a result was not surprising since this residue appears only to be fully conserved in plants and fungi and not in parasites or in some members of the proteobacteria (see Fig. 2). When Ser256, a residue highly conserved across a substantial number of organisms and equivalent to an essential serine implicated in quinone-binding and reduction in Complex II [27,28], was replaced with threonine the mutated alternative oxidase protein was inactive. Such a result suggests that although the hydroxyl group is not critical for activity the steric environment probably is. Arg262, which is part of the HR pair [26], is almost completely conserved throughout all AOX sequences (see Fig. 2) and the question arises as to why lysine is not found at this Fig. 3. Western blot analysis of mutated AOX proteins expressed in S. pombe position. Replacement of Arg262 with lysine, a subtle mutation which mitochondria. Western blots of mitochondria purified from S. pombe expressing wild- retains the positive charge, results in an inactive protein, suggesting type or mutant AOX probed with AOA antibodies. Mitochondrial proteins (15 µg per that the guanidino group rather than its positive charge is critical for lane), loaded on two separate gels (A and B), are as follows: lane1, wild-type AOX; lane 2, Q242N; lane 3, N247Q; lane 4, wild-type AOX; lane 5, Y253F; lane 6, Y253A; lane 7, antimycin-resistant activity. We are aware, however, that in terms of S256T; lane 8, H261A; lane 9, R262K. Numbers on the right of the immunoblot refer to space occupation, the guanidino group extends a much longer distance molecular masses of the standards (in kDa). than the primary amine of lysine and furthermore there are more hydrogen atoms available for hydrogen bonding with arginine in comparison to lysine. Replacement of His261 with alanine has a similar 4. Discussion effect on activity to that observed with R262K, namely total inhibition of antimycin-resistant respiratory activity. As indicated above and as A key challenge in understanding the structure–function relation- can be seen from Fig. 2, His261 is conserved across all AOX sequences ship of the alternative oxidases rests upon the identification of the isolated to date and the lack of activity observed following its ubiquinone-binding site. A detailed knowledge of the nature of this substitution suggests that the imidazole group plays some critical binding site is important not only with respect that it may reveal role in the maintenance of optimal structure/reactivity possibly whether or not there is a common architecture that can be applied to through participating in hydrogen-bonding. quinone-binding sites in general and hence provide an insight into the With respect to Tyr253, a highly conserved residue, interestingly mechanism of binding but perhaps more importantly could assist in mutation to phenylalanine resulted in an active protein thereby the suitable rational development of phytopathogenic fungicides and suggesting that it is the aromatic ring and not H-bonding through the anti-parasitic drugs that are specifically targeted to the alternative hydroxyl group that is important for ubiquinol-binding. This notion is oxidase. As indicated earlier it is now generally recognized that the further confirmed by the finding that replacement of Tyr253 with distribution of the alternative oxidase is substantially wider than alanine, does indeed result in loss of activity (72% inhibition). In some previously thought and is no longer restricted to plants, some fungi senses the fact that the protein was still partially active was an and protists but is also widespread amongst human parasites such as unexpected result since we had assumed that such a substitution trypanosomes, Cryptosporidium parvum, and Blastocystis hominis (see would have resulted in total inhibition due to loss of the aromatic ring Fig. 2). thereby preventing the binding of the quinone head group. Similar We, and others [1,10,11,45,46], have previously suggested that the inhibitory effects have, however, been observed by Nakamura et al. region between helices II and III is the most likely site to be involved in [16] who found that the same mutation in T. vivax recombinant AOX the binding of ubiquinol. This is based upon the following observa- only resulted in a 44% inhibition of the AOX activity. tions namely (1) when any of the alternative oxidase sequences are In order to ascertain that the loss in alternative oxidase activity mapped onto the structure of the Δ9-desaturase protein a hydropho- observed with Gln242, Ser256, His261 and Arg262 mutations is not due bic crevice is revealed which reaches down to the active site of the to the mutated protein failing to associate with the inner mitochondrial protein [10] (2) the crevice contains residues, identified by Berthold membrane western blot analysis, using plant AOA (alternative oxidase- [48] in a mutagenesis screen, that result in an increased resistance to all) antibodies, was performed. Analysis of Fig. 3 reveals three areas of the presumed competitive inhibitor SHAM [50]; (3) these residues interest, namely a band at 32 kDa, which represents AOX monomer, a flank the extremely well conserved HR dyad, part of a potential band at approx 42 kDa, and bands at 76 kDa which represent the hydroquinone-binding motif [26] and (4) the hydrophobic pocket dimeric form of AOX. It should be noted that Fig. 3A and B represent 2 also contains a highly conserved tyrosine [45] which is positioned separate preparations which probably accounts for the variation in proximal to the HR pair. monomer intensity between the various mutants. The band at 42 kDa, In this study we have identified a number of key residues within which we have previously identified as AOX precursor protein [30], this hydrophobic crevice that appear to be critical for alternative does, however, vary considerably amongst the mutants, the reasons for oxidase activity which include Tyr253, Ser256, His261 and Arg262. which are currently unclear. Nevertheless, despite any slight difference Mutation of these residues reveals that the presence of hydroxyl, in signal intensity of the monomer amongst the various mutants, which guanadino, imidazole and aromatic groups, polar and charged may be due to experimental variation, it is readily apparent from Fig. 3 residues in addition to the size of the amino-acid chain are important that all of the mutated AOX proteins are expressed in significant for ubiquinone-binding. amounts and to a similar order of magnitude as wild-type AOX in S. In addition to these amino-acids, analysis of the differences in pombe mitochondria. We can therefore conclude that any difference in sensitivity of the trypanosomal isozymes (Trypanosoma brucei (Tb) activity amongst the various mutants cannot be attributed to a lack of and vivax (Tv)) [16] to the TAO inhibitor, ascofuranone [51], led the mutated forms of AOX failing to associate with S. pombe Crichton [46] to identify two further residues (Gln242 and Asn247) mitochondrial membranes but is more a reflection of the importance which are the only significantly polar residues that are conserved in of that residue for ubiquinol-binding. this hydrophobic region [see ref. 1 for a review]. Gln242 is fully 1938 M.S. Albury et al. / Biochimica et Biophysica Acta 1797 (2010) 1933–1939

Fig. 4. Alignment of the proposed Q binding region of AOX of several fungal plant pathogens compared with Sauromatum guttatum AOX. Alignment constructed using ClustalW, and viewed using SeaView. As before, the Q binding region is defined as residues 236–264 as per Sauromatum guttatum numbering (P22185). Conserved residues are shown in colour — R/K in red, H in blue, Q/N in green, Y in teal and P in yellow. Full species names and accession (UniProt) numbers — Sauromatum guttatum P22185; Magnaporte grisea O93788; Neurospora crassa Q01355; Botrytis cinerea Q8NJ59; Candida albicans AOX1 O93853; Candida albicans AOX2 Q9UV71; Podospora anserina Q9C206; Metarhizium anisopliae Q45N56; Aspergilus niger O74180; Pichia stipidis Q9P414. conserved (see Fig. 2) across all organisms (except for PTOX) and clearly suggest that the two quinol-binding sites are different. Therefore mutation to Asn results in complete loss of activity. Replacement of until more potent and specific inhibitors of the AOX, such as Asn247 with Gln, however, has relatively little effect upon activity and ascofuranone, are developed it is difficult to give a definitive answer Fig. 2 indicates that Asn247 has been replaced by either leucine (Tb) as to whether or not the fungal alternative oxidase does play any or serine (Tv). Interestingly Tv-AOX exhibits an ~8-fold lower KM for significant role in field strobilurin resistance. Nevertheless the fact that ubiquinol-1 than Tb-AOX [16] This difference in affinity for Q fungal pathogens do express an alternative oxidase when treated with correlates with a higher sensitivity of the Tv-AOX to ascofuranone, fungicides is, in our opinion, sufficient evidence to warrant further and explains the higher potency of this drug against T. vivax parasites investigation possibly by developing fungicides that can act on the specifically [16]. It is interesting to speculate that since the residue at ubiquinone-binding site identified in this paper and that could be used position 247 is the only difference between the plant and trypano- alongside strobilurin fungicides. some sequence it may play a key role in ascofuranone sensitivity and therefore represents an attractive residue for future mutagenesis Acknowledgements studies. In particular, introducing a serine or leucine at position 247 in the S. guttatum AOX (to reflect the situation found in the trypanosome KE is grateful to the University of Sussex for receipt of a graduate isozymes) may allow the involvement of this residue in Q binding/ bursary (Research Development Fund). This work was supported by a inhibition to be better assessed and such information may prove grant from the BBSRC (ALM). We would like to thank Dr. Paul Crichton useful in the future rational design of anti-parasitic drugs [5,9,51]. (MRC Mitochondrial Biology Unit, Cambridge) for useful discussions As indicated earlier, the alternative oxidase is also widespread and insight into ubiquinol-binding and Dr Tom Elthon (Univ of amongst fungi and similar to plants is induced when the organism is Nebraska, USA) for the generous gift of AOA monoclonal antibodies. stressed [3,4]. In recent years agricultural fungal control has resulted in the development fungicides specifically targeted to the quinol- References oxidation site (Qo) of the cytochrome bc1 complex [52].One [1] A.L. Moore, M.S. Albury, Further insights into the structure of the alternative important group of Qo site inhibitors that have proved effective in oxidase: from plants to parasites, Biochem. Soc. Trans. 36 (2008) 1022–1026. the control of plant pathogens are the strobilurin fungicides, the most [2] M.S. Albury, C. Elliott, A.L. Moore, Towards a structural elucidation of the widely used one being azoxystrobin [53]. 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