Journal of Experimental Botany, Vol. 53, No. 375, pp. 1833-1836,
August 1, 2002
© 2002 Oxford University Press
Molecular cloning and characterization of plant genes encoding novel peroxisomal molybdoenzymes of the sulphite oxidase family
Received 25 February 2002; Accepted 20 May 2002
2 Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Nara 630-0101, Japan
3 Unité de Nutrition Azotée des Plantes, INRA, 78026 Versailles, France
Abbreviations: Moco, molybdenum cofactor; NR(s), nitrate reductase(s); SOX(s), sulphite oxidase(s); EST(s), expressed sequence tag(s); cyt, cytochrome; FAD, flavin adenine dinucleotide; GFP, green fluorescent protein.
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Abstract |
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The Arabidopsis AtMCP and rice OsMCP genes which encode proteins highly homologous to molybdoenzymes of the sulphite oxidase family were isolated and characterized. Both proteins seemed to possess only a molybdenum cofactor as the redox centre, unlike all the other eukaryotic molybdoenzymes. Putative MCP orthologues were identified in 17 plant species, indicating that Mo possess only a molybdenum cofactor as the redox centre, unlike all the other eukaryotic molybdoenzymes. Putative MCP orthologues were identified in 17 plant species, indicating that MCPs are widely distributed over the plant kingdom. An analysis using a green fluorescent protein fusion showed that AtMCP possesses a peroxisomal targeting signal at its C-terminus. Putative peroxisomal targeting signals were also found in all plant MCPs, suggesting the existence of a new redox pathway in this organelle.
Key words: Key words: Expressed sequence tag, green fluorescent protein, molybdenum cofactor, nitrate reductase, peroxisomal targeting signal, redox reaction.
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Molybdenum cofactor (Moco)-containing enzymes catalyse hydroxylation or oxygen atom transfer on various carbon-, sulphur- and nitrogen-compounds in a two-electron transfer reaction mediated by the Moco. Based on amino acid sequence similarities, Moco-containing enzymes are classified into four groups: the sulphite oxidase (SOX), DMSO reductase, xanthine oxidase, and aldehyde ferredoxin oxidoreductase families (Kisker et al., 1997a). Most of the Moco-containing enzymes have additional redox centres, such as iron–sulphur centres, haems and/or FADs, with the exception of Rhodobacter DMSO reductase and bacterial SOX family proteins (Kappler et al., 2000).
SOX, which makes up the SOX family with assimilatory nitrate reductases (NRs), has been characterized in animals (Kisker et al., 1997b; Neame and Barber, 1989; Garrett and Rajagopalan, 1994) and bacteria (Kappler et al., 2000). Eukaryotic SOX seems to be a dimeric enzyme and each monomer houses two prosthetic groups: a cyt b5 and a Moco. Animal SOX is located in the mitochondrial intermembrane space where it oxidizes sulphite to sulphate, the last step of sulphur-containing amino acids’ degradation. Assimilatory NRs catalyse the reduction of nitrate into nitrite and have three redox centres, namely FAD, cyt b5 and Moco, associated with three separate domains which transfer, in this order, electrons from NAD(P)H to nitrate.
In plants, several genes encoding Moco-containing enzymes have so far been identified and characterized (i.e. NRs, xanthine dehydrogenases and aldehyde oxidases). Another molybdoenzyme whose existence in plants has been discussed is SOX (Mendel and Schwarz, 1999; Jolivet et al., 1995). Though plant SOX enzymatic activities were partially characterized (Jolivet et al., 1995; Ganai et al., 1997), it is unclear whether this plant SOX activity derives from a molybdoenzyme structurally similar to animal or bacterial SOXs. To address this question, plant DNA databases were searched for genes encoding putative SOX homologues. The cloning and characterization of two plant cDNAs, AtMCP and OsMCP, from Arabidopsis and rice, respectively, which code for proteins highly homologous to the Moco-containing domain of the SOX protein family is reported here. Their possible biological function is also discussed.
Arabidopsis EST clones were obtained from the Arabidopsis Biological Resource Center, Ohio State University. The template for 5'RACE-PCR was prepared using the MarathonTM cDNA Amplification Kit (CLONTECH) and total RNA isolated from green tissues of Arabidopsis (Columbia ecotype). 5'RACE-PCR was performed according to a provided protocol using atmcp-1, 5'-GCGTTGACCTTGAGAGAAGG-3' and atmcp-2, 5'-AAGATG AGACTAAGGCCGAGCGG-3', and the PCR fragments obtained were sequenced directly. To check whether the fused sequence is actually derived from the AtMCP gene, a full-length cDNA was amplified by PCR using primers atmcp-g1, 5'-TGTTTGG GCTCAATTTGGGC-3', which is derived from the sequence just downstream of the putative transcription start site; and atmcp-6, 5'-CTGCAGGTCTACAAGTTAGAGTGG-3'. The putative AtMCP transcription start site was determined by primer extension as described below.
A homologous rice EST clone was also identified. To determine the nucleotide sequence of this cDNA, 5' and 3'RACE-PCR were performed with template from Oryza sativa cv. Nipponbare and the following gene-specific primers: osmcp-1, 5'-TCTTGGAAATGC AACATGGG-3'; osmcp-2, 5'-GTGGGTATCCATGATCACGG-3'; osmcp-3, 5'-CGAAATGAACGGAGAGACCC-3'; osmcp-4, 5'-GAGCGTGCGCCAATTACTCC-3'. The PCR fragments obtained were cloned into a vector and sequenced. Then, a full-length cDNA was obtained by 5'RACE-PCR with primer osmcp-9: 5'-TTTTT TTTTTTTTAGGAGCACAAGAACTGC-3', designed at the 3'-end of the cDNA. Amplified PCR fragments were sequenced directly on both strands.
Primer extension reaction was carried out using 2 µg of total RNA prepared from flower tissue and the Texas Red labelled atmcp-1 primer. A sequence ladder used as molecular size markers was generated with the same primer and the EST clone plasmid (GenBank Acc. No. T22091). Signals were detected using an automated DNA sequencer (Model SQ5500, Hitachi).
pGFP-GHSNL was constructed using the vector pmGFP(GA)5II (provided by Pius Spielhofer). The DNA fragment corresponding to the last five amino acids of AtMCP was prepared by annealing a pair of oligonucleotides: atmcp-C2-S, 5'-TCGAGGCCACTCTAACTT GTAG-3'; and atmcp-C2-AS, 5'-CTACAAGTTAGAGTGGCC-3'. The DNA fragment was inserted into pmGFP(GA)5II which was digested with both Sal I and Sma I, and the nucleotide sequence was confirmed by sequencing.
Tobacco (Nicotiana tabacum L. cv. Bright Yellow 2) suspension-cultured cells were transiently transformed by microprojectile bombardment with 1 µg of plasmid and a Biolistic Particle Delivery System (Bio-Rad). After bombardment, the cells were left for 16 h at 27 °C, and then observed under a fluorescence microscope (Diaphot300, Nikon) with a filter block (B-2E/C, Nikon).
In an attempt to examine if higher plants have a SOX homologue, translated DNA databases were searched with the amino acid sequence of the chicken SOX (SwissProt Acc. No. P07850) as a query. As expected, sequences corresponding to NR genes were found as showing high homology with both the Moco- and the cyt b5-containing domains of chicken SOX. But the best homology scores were obtained from an Arabidopsis EST (GenBank Acc. No. T22091) and a rice EST (GenBank Acc. No. AA751890) sequence. The amino acid sequences deduced from these two ESTs were indeed highly homologous to the Moco domain of chicken SOX and were different from the known NR sequences. Based on the sequences of these ESTs, 5' and 3' RACE reactions were performed. The obtained cDNA sequences from Arabidopsis and rice were designated as AtMCP (Arabidopsis thaliana Moco-containing protein, DDBJ Acc. No. AB071965) and OsMCP (Oryza sativa Moco-containing protein, DDBJ Acc. No. AB071966), respectively.
The full-length AtMCP cDNA and the OsMCP cDNA encode a 43.3 kDa and a 43.7 kDa protein, respectively (Fig. 1A). A search in the complete Arabidopsis genome sequence indicated that AtMCP is a single-copy gene consisting of 12 exons. These results indicate that the amplified sequence is indeed derived from the AtMCP gene. This was also confirmed by PCR amplification of a full-length AtMCP cDNA containing the transcription start site, which was determined by the primer extension analysis (data not shown). However, all the sequences corresponding to AtMCP, including predicted transcribed sequences and EST sequences, deposited so far in the databases have shorter 5' sequences than the cDNA sequence reported here.
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The amino acid sequences of AtMCP and OsMCP are very homologous (76% of identity) and are closer to the protein sequence of the Moco-containing domain of chicken SOX (Neame and Barber, 1989) (about 47% identical) than to that of Arabidopsis NR (GenPept Acc. No. AAG51627, about 32% identical). Based on the crystal structure of chicken liver SOX, this Moco-containing domain consists in fact of two subdomains, the first one (domain II) binds the Moco and the C-terminal one (domain III) is mainly involved in the formation of a dimer structure (Kisker et al., 1997b). This domain III is also conserved in the MCP sequence which suggests that MCP may be a dimeric protein in plants. In MCPs, 10 out of the 11 amino acid residues that are known to be involved in Moco binding in chicken SOX (Kisker et al., 1997b) are conserved (Fig. 1A). Furthermore, five out of the six amino acid residues involved in the binding of the sulphate oxygens in chicken SOX (Kisker et al., 1997b) are conserved in MCPs (Fig. 1A). On the other hand, only three out of these six conserved residues are found in the Arabidopsis NR sequence (GenPept Acc. No. AAG51627). These results suggest that MCP proteins contain Moco and catalyse oxygen atom transfer to or from a substrate structurally similar to sulphite or sulphate. One possibility is that MCPs are plant SOXs. Indeed SOX activity has been detected in plants (Jolivet et al., 1995; Ganai et al., 1997) and the closest plant sequences to animal SOX are the MCP sequences. Whereas typical animal SOXs have both a Moco and a cyt b5 domain, the putative MCPs seem to possess only a Moco domain (Fig. 1B). This makes the MCPs the only eukaryotic Moco-containing proteins which do not have another redox domain linked to the Moco domain. These results also indicated that MCPs could be assigned as a third and new member of the eukaryotic SOX family, in addition to SOX itself and to plant NR. The possibility that the obtained nucleotide sequences lack some 5' sequence was excluded by the result of the primer extension analysis which showed that there is no coding sequence upstream of the Moco domain. This was also supported by the three in-frame stop codons at the 5' UTR of AtMCP.
BLAST similarity searches of ESTs (dbEST, January 2002 release) with the AtMCP amino acid sequence as a query identified more than 90 ESTs in other plant species than Arabidopsis and rice. These ESTs were found to be highly homologous to AtMCP at the amino acid level and are derived from 15 plant species including monocots, dicots, a moss and an alga (Table 1). This result indicates that putative MCP orthologues are widely distributed over the plant kingdom. Several ESTs from 11 plant species covered the MCP C-terminal regions, and all of their C-terminal tripeptide sequences, as well as those of AtMCP and OsMCP, fitted well to one type of the peroxisomal targeting signal (PTS1) (Table 1). PTS1 is a unique tripeptide sequence found in the C-terminus of peroxisomal proteins with proposed consensus motifs, [A/C/P/S]-[K/R]-[I/L/M] (Hayashi et al., 1997) and [A/C/G/S/T]-[H/K/L/N]-[I/L/M/Y] (Mullen et al., 1997). To examine whether MCPs are actually targeted to peroxisomes, a fusion protein, GFP-GHSNL, comprised the C-terminal pentapeptides (-GHSNL) of AtMCP fused to the C-terminal end of the green fluorescent protein (GFP) was transiently expressed in tobacco suspension-cultured cells (Fig. 2). The fluorescence pattern of the GFP-GHSNL was similar to the peroxisomal signals obtained with a plasmid, pMAT-SGFP-H1, which expresses a protein targeted to peroxisomes (Mano et al., 1999). These results indicate that MCPs are peroxisomal proteins and their targeting are mediated by PTS1 type signals. The putative function of MCPs in plants still remains an open question. The peroxisomal localization of the MCPs does not exclude a participation of these proteins in sulphite detoxification. Alternatively, as sulphite and nitrite are structurally similar ions, one cannot exclude a participation of the MCPs in nitrite detoxification by oxidation into nitrate (Meyer and Stoehr, 2002). Indeed, the other plant enzyme which uses sulphite as substrate, the sulphite reductase, is also capable of reducing nitrite as well as sulphite (Krueger and Siegel, 1982). Moreover, nitrite rarely accumulates to a high level in plants and it has been suggested that plants are able to oxidize nitrite into nitrate (Aslam et al., 1987; Meyer and Stoehr, 2002). The fact that metabolism in peroxisomes is essentially of the oxidative type may also suggest a participation of the MCPs in oxidative detoxification of such toxic anions.
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Acknowledgements |
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We would like to thank the Arabidopsis Biological Resource Center for supplying the Arabidopsis EST clone, Dr Shoji Mano and Dr Mikio Nishimura for providing the pMAT-SGFP-H1 vector, and Dr Pius Spielhofer and Dr Nam-Hai Chua for providing the pmGFP(GA)5II vector. This work was partly supported by a grant for Research for the Future Program (JSPS-RFTF 00L01604) from the Japan Society for the Promotion of Science.
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