(Received for publication, August 25, 1994)
From the
Most organisms appear to have a molybdenum cofactor consisting of a complex of molybdenum and a pterin derivative. Very little is known about molybdenum cofactor biosynthesis in plants or other eukaryotes, because the instability of the cofactor and its precursors makes it difficult to analyze this pathway. We have isolated two cDNA clones from the higher plant Arabidopsis thaliana encoding genes involved in early steps of molybdenum cofactor biosynthesis. The cDNAs were obtained by functional complementation of two Escherichia coli mutants deficient in single steps of molybdenum cofactor biosynthesis. The two cDNAs, designated Cnx2 and Cnx3, encode proteins of 43 and 30 kDa, respectively. They have significant identity to the E. coli genes, moaA and moaC, involved in molybdenum cofactor biosynthesis. Both genes have N-terminal extensions that resemble targeting signals for the chloroplasts or the mitochondria. Import studies with the translated proteins and purified mitochondria and chloroplasts did not show import of these proteins to either of these organelles. Northern analysis show that Cnx2 is expressed in all organs and strongest in roots. Cnx3 is not expressed in abundant levels in any tissue but roots. For both genes there is no detectable difference in the expression level from plants grown with nitrate or with ammonium. The Cnx2 gene has been mapped to chromosome II. Southern analysis suggests that both genes exist as single copies in the genome.
All molybdoenzymes studied to date, except for
molybdo-dinitrogenases, contain a common molybdenum cofactor. This
molybdenum cofactor (MoCo) ()is essential for the activity
of several key enzymes like nitrate reductase, xanthine dehydrogenase,
sulfite oxidase, aldehyde oxidase, and formate
dehydrogenase(1) . These enzymes are important in both
prokaryotic and eukaryotic pathways such as nitrogen assimilation,
sulfur and purine metabolism, and hormone biosynthesis.
The basic structure of the molybdenum cofactor appears to be the same in all organisms investigated(2) . The molybdenum cofactor consists of a 6-alkyl pterin (named molybdopterin) proposed to be complexed with molybdenum through the sulfur atoms of a dithiolene moeity on its 6-alkyl side chain(3) . In plants the MoCo is thought to be in the pterin-base form, but in some organisms such as Escherichia coli MoCo exists in a nucleotide form, where a guanine dinucleotide is linked to the 4-carbon side chain of the moeity(2) . Fig. 1shows the proposed structure of MoCo in plants and in E. coli.
Figure 1: The structure of MoCo in plants (A) and in E. coli (B). Structure is modeled after that done by Rajagopalan and Johnson(2) .
Very little information is
available on MoCo biosynthesis in plants or other eukaryotes. The
instability of the cofactor and its precursors makes it difficult to
establish the biosynthetic pathway by direct detection and analysis of
the intermediates. The isolation and analysis of mutants affected in
MoCo biosynthesis have made it possible to classify them into several
complementation groups corresponding to different steps in MoCo
biosynthesis. Chlorate resistance has been the basis for selecting MoCo
mutants in bacteria, fungi, algae, and higher plants (for reviews see (4) ). The mutants are pleiotropically defective in nitrate
reductase and other molybdoenzyme activities. In plants, MoCo-deficient
mutants have been classified into six different complementation groups (cnxA-cnxF), indicating that at least 6 genes are
involved in MoCo biosynthesis (see (5) and (6) for
reviews). The genes and their precise functions in MoCo biosynthesis
are not known. In Nicotiana plumbaginifolia, the cnxA complementation group shows a characteristic different from the
other groups; their phenotype can be partially restored when grown on
high levels of molybdate(7, 8) . This led to the
hypothesis that the CnxA gene product is involved in the
insertion of molybdenum in the molybdopterin(7) . MoCo
deficiency in plants leads to the inability to use nitrate as a
nitrogen source due to a lack of active nitrate reductase enzyme. N. plumbaginifolia MoCo mutants can be partially rescued
either by grafting, where they show a chlorotic and wilty phenotype, or
by growth in vitro on a reduced N source, where they show
developmental abnormalities such as reduced leaf size and long
internodes(9) . The wilty phenotype is due to a defect in
abscisic acid biosynthesis, linked to the absence of the MoCo enzyme
aldehyde oxidase()(10) .
Several cases of MoCo deficiency have been reported in humans. It causes mental retardation and other severe symptoms and leads to death in infancy(11) . By co-culturing cells from affected individuals, it has been possible to identify two complementation groups in humans (11) . No treatment is yet possible, and the diagnostics and prenatal testing are difficult. MoCo mutants have been studied in Drosophila melanogaster, where such mutants are easily identified by eye color variations. This variation is a result of reduced or deficient xanthine dehydrogenase(12) . Recently a gene involved in molybdenum cofactor biosynthesis has been isolated from Drosophila(13) .
MoCo and its biosynthesis have been studied extensively in E. coli (for a review, see (2) ). Five loci involved in the synthesis of a mature MoCo have been identified by screening for chlorate resistance. Each locus encodes one or more enzymes in the MoCo biosynthetic pathway. Two loci, moa and moe, are required for early steps of MoCo biosynthesis: the synthesis of molybdopterin. The sequence has been determined for both loci(14, 15) . The moa locus contains five genes in an operon (moaA-moaE). The moaA-moaC gene products are thought to be involved in molybdopterin precursor production, while the moaD and moaE encode two proteins forming a heteromeric ``converting factor'' that is responsible for the conversion of the precursor into molybdopterin(15, 16, 17) . The moe locus is probably involved in the sulfur donation to the converting factor(18) .
The aim of this study was to obtain information on MoCo biosynthesis in plants by cloning genes involved in its biosynthesis. As the basic structure of MoCo is probably the same in all organisms, its biosynthesis might share common steps as well. Our approach was to complement E. coli MoCo mutants with plant cDNAs by selecting for restored ability of an E. coli MoCo mutant to use nitrate as the terminal electron acceptor in anaerobic conditions (restored nitrate reductase activity). Here we report the isolation, characterization, and functional expression of two genes involved in early steps of MoCo biosynthesis in Arabidopsis thaliana. These cDNAs specifically supressed the E. coli moaA and moaC mutations.
The E. coli mutant KB2066 (affected in gene C in
the moa locus) was transformed with the cDNA plasmid library
in the same manner. 8 10
total transformants were
obtained upon four electroporations. After 5 days of growth under
selective conditions two groups of colonies appeared: small and large
(29 small colonies and 55 large colonies). Plasmids from six small
colonies and 12 large colonies were prepared, and upon retransformation
of the mutant only the plasmids of the group giving rise to small
colonies were able to complement the mutant. The group consisting of
large colonies were probably revertants. The six complementing plasmids
had approximately the same size insert and similar DNA restriction
pattern.
Plasmid p39A5 complementing the KB2039 mutant and plasmid p66A4 complementing the KB2066 mutant were selected for further analysis.
Figure 2:
In vivo nitrate reductase assays
of the non complemented and the complemented moa mutants. The
mutants KB2039 and KB2066 and the mutants containing the Arabidopsis cDNAs: p39A5 (Cnx2) and p66A4 (Cnx3), respectively, were used for the assay. The wild type E. coli strain MC4100 was used as control. Each value is the
mean of three independent experiments. The NO produced are expressed in nmol/min/10
cells, and for
each experiment the percentage of the wild type level is
indicated.
The lower level of NO produced
by the p66A4 complemented moaC mutant, and thereby the lower
level of nitrate reductase activity, when compared to the wild type,
may explain the slow growth of the complemented moaC mutant on
solid selection medium in the original complementation experiment,
where only small colonies where obtained. It might simply reflect poor
complementation by the Arabidopsis protein, or it could be a
question of optimizing expression of the construct. We know that the
coding region of this clone is not in frame with the ATG in the
expression vector, so, upon expression under the lac promoter,
the proper translation of the product must arise from translation
re-initiation probably at the first ATG in the clone. Furthermore,
p66A4 is not a full-length clone (see below), but the missing part is
in the N-terminal extension that is not present in the corresponding E. coli gene.
Figure 3: Nucleotide sequence and the deduced amino acid translation of the Arabidopsis cDNAs: Cnx2 from plasmid p39A5 (A) and Cnx3 from plasmid p66A4 (B). Numbers on the left and on the right correspond to the nucleotide base pair and amino acid positions, respectively. Sequences corresponding to the primers used for amplification of the 5` ends are underlined. The 5` sequences provided from the RACE clones are shown in bold.
We have named this cDNA Cnx2 according to the guidelines of Caboche et al.(34) . The mnemonic cnx (cofactor of nitrate reductase and xanthine dehydrogenase) is the same as used for plant MoCo-deficient mutants. Even though this is the first Cnx gene cloned from plants, it is given the number 2, because Cnx1 is assigned to the molybdenum-repairable locus (CnxA) once it is identified(34) .
Figure 4: Southern blot analysis of Arabidopsis genomic DNA. 2 µg of genomic DNA was digested with each of the restriction enzymes indicated on the figure. In A the Cnx2 cDNA was used as probe, and in B the Cnx3 cDNA was used as probe.
Figure 5:
Northern blot analysis of total RNA from
different plant tissues. The lanes were loaded with approximately 15
µg of total RNA from leaves (L), roots (R),
siliques (S), flowers (F), cell suspension culture (C), and from whole plantlets grown in vitro with
KNO (+) or with NH
-succinate (-).
The same filter was probed with three different probes: the entire Cnx2 cDNA, the entire Cnx3 cDNA, or the Arabidopsis Nia2 cDNA (nitrate reductase
gene).
Figure 6: Peptide alignment of Cnx2 and Cnx3 from Arabidopsis with their E. coli homologs. A, alignment of the deduced amino acid sequence of Cnx2 from Arabidopsis and of MoaA from E. coli. B, alignment of the deduced amino acid sequence of Cnx3 from Arabidopsis and MoaC from E. coli. Verticallines indicate identical residues, and doublepoints indicate conservative changes. Sequences were obtained from GenBank and have the following accession numbers: P30745 and P30747 for moaA and moaC, respectively.
Figure 7: Multiple alignment of the predicted amino acid sequence of Cnx2 with related proteins. The following proteins are aligned with Cnx2: MoaA from E. coli (accession number P30745), Nifb from Anabaena sp. (accession number J05111), Fixz from Rhizobium leguminosarum (accession number P07748), PQQ synthesis protein III from Acinetobacter calcoaceticus (accession number X06452) and Fdhc-orf, an open reading frame from Methanobacterium formicium (accession number M64798). The numbering is according to the Arabidopsis Cnx2 peptide sequence. Darkgrayshadedboxes represent sequence identity, and lightgrayshadedboxes represent at least 3 identities in the alignment. The alignment was made with Pileup (GCG program) and adjusted by hand.
Alignment of the E. coli MoaA and the Arabidopsis Cnx2 sequences reveals that the Arabisopsis cDNA has a 60-amino acid extension in the N-terminal region of the peptide translation. This N-terminal extension has some characteristics of transit peptides for either the chloroplast or for the mitochondria: a high percentage of hydroxylated amino acids and a low level of acidic amino acids(35, 36) . To analyze the function of the N-terminal extension, import studies with purified chloroplasts and mitochondria were performed. The results are described in a separate section below.
Figure 8: Chloroplast and mitochondrial import studies with p39A5 (Cnx2) and p66A4 (Cnx3) translation products. The positive controls used are psaD and superoxide dismutase 3 (SOD3) translation products, respectively localized in the chloroplast and in the mitochondria. A shows the SDS-PAGE fluorography of the chloroplast import experiment, and B shows the SDS-PAGE fluorography of the mitochondrial import experiment. The tlanes are loaded with 1 µl of the translation mix, the c and mlanes are loaded with import reaction (corresponding to one-sixth of the total import reaction). The planes are loaded with protease-treated import reaction (corresponding to one-sixth of the total import reaction).
We have cloned and characterized two Arabidopsis cDNAs able to restore nitrate-dependent anaerobic growth in two E. coli mutants affected in early steps of MoCo biosynthesis. The two MoCo-deficient, and therefore NR-deficient, E. coli mutants had at least partially restored nitrate reductase activity when complemented with the cDNAs. Both cDNA peptide translations have significant homology to the E. coli molybdenum biosynthetic genes moaA and moaC, respectively. On the basis of their function and sequence similarities, we suggest that the Arabidopsis cDNAs correspond to enzymes involved in MoCo biosynthesis. To our knowledge these are the first characterized cDNAs encoding enzymes involved in MoCo biosynthesis in plants. Very little information is available on this pathway in any eukaryote. Cloning the plant cDNAs by functional complementation of E. coli mutants indicates that some steps of the MoCo biosynthesis are identical in prokaryotes and plants. With the gene sequences from Arabidopsis and E. coli, it might now be possible, by a PCR approach, to clone the human genes to obtain diagnostic tools for MoCo deficiency in humans. It would be interesting to see if the conservation between these early steps of MoCo biosynthesis is true for all eukaryotes.
Recently a gene involved in MoCo biosynthesis has been cloned from Drosophila. This gene appears to encode a multifunctional
protein because different regions show homology to three different MoCo
genes from E. coli(13) . A similar gene has been
identified in plants. ()Cnx2 and Cnx3 appear to encode monofunctional proteins, although we cannot
exclude the possibility that the N-terminal extensions could encode
other functions. The N termini do not show homology with the sequenced E. coli MoCo genes or any other gene upon a search in the
GenBank data base. In the purine biosynthetic pathway, we find
monofunctional enzymes in E. coli and in plants, while the
homologous enzymes are multifuntional in other eukaryotes(29) .
It remains to be shown if MoCo biosynthesis in plants is dominated by
monofunctional genes as in E. coli.
We have tried to complement two other E. coli MoCo mutants, affected in the moa locus gene D and E, respectively. We failed to complement these mutations with plant cDNAs. The reason for the unsuccessful complementation could be the fact that these proteins, encoded by moaD and moaE, are part of a heteromeric complex(17) . This means that the proteins not only need the proper enzymatic function to complement, they also need a specific structure to fit into the E. coli complex.
The Cnx2 and Cnx3 products probably have the same function as the E. coli proteins encoded by moaA and moaC, which are suggested to be responsible for molybdopterin precursor production(16, 17) . The precursor is a 6-alkyl pterin with a 4-carbon phosphorylated side chain. It has been shown that the phosphate is bound in diester linkage to C-2` and C-4` of the side chain to form a six-membered ring and that the precursor does not contain any of the sulfurs present in the mature MoCo(37, 38) . It is believed that molybdopterin synthesis is different from the synthesis of other known pterins like folic acid and tetrahydrobiopterin, since their precursor, dihydroneopterin triphosphate, has a 3-carbon alkyl side chain(37) . The substrate for the molybdopterin precursor synthesis is not yet defined, but studies with labeled guanosine in E. coli indicate that the substrate may be a derivative of GTP(2) .
Some of the sequence similarities that were established for the MoaA peptide (15) can also be found for the Cnx2 peptide. Cnx2 has regions of similarity with guanine-binding proteins. Moreover, similarity can be established between Cnx2 and a protein in PQQ synthesis, a cofactor whose structure resembles a pterin moeity. The homology of Cnx2 to NifB and FixZ proteins was not expected as these proteins are involved in early steps of the iron-molybdenum cofactor biosynthesis:; the cofactor of dinitrogenase. The two cofactors have different structures; the iron-molybdenum cofactor is homocitrate-based, while the MoCo is a pterin derivative. The similarity that was established between the moaA gene product from E. coli and the molybdoenzyme formate dehydrogenase-H (15) cannot be found to the plant Cnx2 gene product.
An interesting feature of the two plant proteins is their N-terminal extension when compared to the E. coli homologs. They have some of the characteristics of transit peptides for the chloroplasts and mitochondria. The fact that guanine, a likely precursor in MoCo biosynthesis, probably is made in the plastids(39) , supports a possible plastid localization of early steps of MoCo biosynthesis in plants. Although there are differences between targeting signals for chloroplast and mitochondrial proteins(35) , these are not obvious and it was not possible for us to predict if the putative transit peptides are for the chloroplasts or the mitochondria. We decided therefore to do import studies with both organelles. The inabilty to demonstrate import of these peptides to chloroplasts or mitochondria in our experiments are not necessarily conclusive. The Cnx2 and Cnx3 proteins might need factors for importation that are not present in standard in vitro import experiments. The Cnx3 clone might not contain all the signals for import and processing as it was not complete in length. Other possible roles for the N-terminal extensions should not be ignored; they could, for example, encode other enzyme activities so the genes simply encode bifunctional enzymes as mentioned previously. The N-terminal extensions could also be needed for specific plant structural features of these enzymes.
Expression analysis of these genes show that the Cnx2 gene is expressed in all organs and with the strongest expression in roots and cell suspension culture. Cnx3 is not strongly expressed in any tissue except in roots and in suspension culture cells. It is surprising that we detect nearly no Cnx3 transcript in leaves. This observation suggests that MoCo biosynthesis has no direct links with photosynthesis, this is supported by the fact that both genes are expressed in etiolated seedlings (results not shown). The plant molybdo-enzyme nitrate reductase is active in leaves, the major site of nitrate reduction. The low expression of Cnx2 and Cnx3 in leaves suggest that nitrate reductase is probably not a major sink for MoCo utilization. It remains to be understood why there is a high level of expression of both genes in roots. This may reflect a MoCo requirement for another molybdo-enzyme located in the roots. For both genes there is no detectable difference in the mRNA level from plants grown with nitrate or with ammonium. These experiments were done to investigate if the mRNA levels of these genes are regulated by some of the same factors as nitrate reductase. The fact that other key enzymes in the plant need MoCo as well may explain why the nitrogen source of the plant have no influence on the two genes' expression level.
Further studies of these genes will include complementation experiments to determine which of the six complementation groups of N. plumbaginifolia, if any, the Cnx2 and Cnx3 genes correspond to. It will also be interesting to know were in the cell these early steps of MoCo biosynthesis take place. Therefore we intend to use other approaches to establish the location of the two proteins: by immunolocalization studies and by analyzing transgenic plants containing fusions of a reporter gene with the putative targeting signals.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z48046 [GenBank](Cnx3) and Z48047 [GenBank](Cnx2).