Isolation of Two Arabidopsis cDNAs Involved in Early Steps of Molybdenum Cofactor Biosynthesis by Functional Complementation of Escherichia coli Mutants

Hoff, Tine; Schnorr, Kirk; Meyer, Christian; Caboche, Michel

doi:10.1074/jbc.270.11.6100

Cited by 67 publications

(52 citation statements)

References 35 publications

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“…For its bacterial homolog (MoaA protein in E. coli and Staphylococcus aureus), the complex reaction mechanism has been deciphered in detail (33,34). As the plant gene cnx2 (35) and the human gene MOCS1A (26) are able to functionally complement their bacterial counterparts, one can assume that the reaction mechanism is likely to occur similarly in eukaryotes. The N-terminal [4Fe-4S] cluster binds SAM and carries out the reductive cleavage of SAM to generate the 5Ј-deoxyadenosyl radical, which subsequently initiates the transformation of 5Ј-GTP bound through the C-terminal [4Fe-4S] cluster.…”

Section: Biosynthesis Step 1: Conversion Of Gtp To Cpmpmentioning

confidence: 99%

The Molybdenum Cofactor

Mendel

2013

Journal of Biological Chemistry

235

181

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The transition element molybdenum needs to be complexed by a special cofactor to gain catalytic activity. Molybdenum is bound to a unique pterin, thus forming the molybdenum cofactor (Moco), which, in different variants, is the active compound at the catalytic site of all molybdenum-containing enzymes in nature, except bacterial molybdenum nitrogenase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also require iron, ATP, and copper. After its synthesis, Moco is distributed, involving Mocobinding proteins. A deficiency in the biosynthesis of Moco has lethal consequences for the respective organisms.The transition element molybdenum is an essential micronutrient for microorganisms, plants, and animals (1). Surprisingly, molybdenum itself is catalytically inactive in biological systems until it is complexed by a special scaffold (2). One type of scaffold is the ubiquitous pterin-based molybdenum cofactor (Moco), 2 which, in different variants, forms part of the active centers of all molybdenum enzymes in living organisms, except one. This exception is bacterial nitrogenase, which harbors the other type of cofactor, namely the Fe-S cluster-based FeMo cofactor, which is found only once in nature (for details, compare the minireview of Hu and Ribbe (62) in this thematic series). Molybdenum belongs to the group of trace elements, i.e. the organism needs it only in minute amounts; however, unavailability of molybdenum is lethal for the organism. Molybdenum is very abundant in the oceans in the form of the molybdate anion (3). In soils, the molybdate anion is the only form of molybdenum that is available for bacteria, plants, and fungi. More than 50 enzymes are known to be molybdenumdependent. The vast majority of them are found in bacteria, whereas only seven have been identified in eukaryotes (4, 5). It is somewhat surprising that not all organisms need molybdenum. The commonly used eukaryotic model organism yeast plays no role in molybdenum research, as Saccharomyces cerevisiae does not contain either molybdenum enzymes or the Moco biosynthesis pathway. Also Schizosaccharomyces pombe does not use molybdenum, whereas Pichia pastoris needs molybdenum. Genome-wide database analyses revealed a significant number of bacteria and unicellular eukaryotes that do not need molybdenum, whereas all multicellular eukaryotes are dependent on molybdenum (6). In addition, mainly anaerobic archaea and some bacteria are molybdenum-independent, but they require tungsten for their growth (7). In the periodic table of elements, tungsten lies directly below molybdenum and features chemical properties similar to molybdenum.Molybdenum metabolism is tightly connected to Fe-S cluster synthesis in that some of the molybdenum enzymes and Moco biosynthesis itself depend on Fe-S enzymes and on a mitochondrial transporter that is known to be crucial for the maturation of cytosolic Fe-S proteins. Moreover, Moco biosynthesis recruits mechanisms previously known from Fe-S cluster synthe...

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Section: Biosynthesis Step 1: Conversion Of Gtp To Cpmpmentioning

confidence: 99%

The Molybdenum Cofactor

Mendel

2013

Journal of Biological Chemistry

235

181

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“…The two E. coli proteins are specified by two separate cistrons, moaA and moaC (13,14,20,25,26) and in the higher plant, Nicotiana tabaccum, by unlinked genes (27). Both of these protein activities are implicated in the synthesis of molybdopterin precursor Z from guanosine or a guanosine derivative.…”

Section: Fig 4 Dna Sequence Analysis Of Cnx Mutations and Inferred mentioning

confidence: 99%

The Aspergillus nidulans cnxABC Locus Is a Single Gene Encoding Two Catalytic Domains Required for Synthesis of Precursor Z, an Intermediate in Molybdenum Cofactor Biosynthesis

Unkles

Smith

Kanan

et al. 1997

Journal of Biological Chemistry

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The Aspergillus nidulans complex locus, cnxABC, has been shown to be required for the synthesis of precursor Z, an intermediate in the molybdopterin cofactor pathway. The locus was isolated by chromosome walking a physical distance of 65-kilobase pairs from the brlA gene and defines a single transcript that encodes, most likely, a difunctional protein with two catalytic domains, CNXA and CNXC. Mutations (cnxA) affecting the CNXA domain, mutants (cnxC) in the CNXC domain, and frameshift (cnxB) mutants disrupting both domains have greatly reduced levels of precursor Z compared with the wild type. The CNXA domain is similar at the amino acid level to the Escherichia coli moaA gene product, while CNXC is similar to the E. coli moaC product, with both E. coli products encoded by different cistrons. In the wild type, precursor Z levels are 3-4 times higher in nitrate-grown cells than in those grown on ammonium, and there is an approximately parallel increase in the 2.4-kilobase pair transcript following growth on nitrate, suggesting nitrate induction of this early section of the pathway. Analysis of the deduced amino acid sequence of several mutants has identified residues critical for the function of the protein. In the CNXA section of the protein, insertion of three amino acid residues into a domain thought to bind an iron-sulfur cofactor leads to a null phenotype as judged by complete loss of activity of the molybdoenzyme, nitrate reductase. More specifically, a mutant has been characterized in which tyrosine replaces cysteine 345, one of several cysteine residues probably involved in binding the cofactor. This supports the proposition that these residues play an essential catalytic role. An insertion of seven amino acids between residues valine 139 and serine 140, leads to a temperature-sensitive phenotype, suggesting a conformational change affecting the catalytic activity of the CNXA region only. A single base pair deletion leading to an in frame stop codon in the CNXC region, which causes a null phenotype, effectively deletes the last 20 amino acid residues of the protein, indicating that these residues are necessary for catalytic function.More than 3 decades ago, Pateman, Cove, and co-workers (see Refs. 1 and 2; reviewed in Refs. 3-6) first isolated mutants defective in the synthesis of the molybdenum pterin cofactor. Using the lower eukaryotic model, the ascomycetous fungus Aspergillus nidulans, they isolated a class of chlorate-resistant mutants, which were unable to utilize either nitrate or purines such as adenine, hypoxanthine, and xanthine as sole sources of nitrogen. This inability to grow on nitrate and hypoxanthine was due to the pleiotropic loss of NADPH-nitrate reductase and NADH-xanthine dehydrogenase (purine hydroxylase I) activity, respectively. They speculated that such mutants were defective in the synthesis of a cofactor common to both nitrate reductase and xanthine dehydrogenase and accordingly designated the mutants cnx (common component for nitrate reductase and xanthine dehydrogenase). Fiv...

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“…In the first step, 59-GTP is converted to precursor Z , which was recently renamed cyclic pyranopterin monophosphate (cPMP) (Schwarz, 2005). This reaction is catalyzed by two proteins, the cofactor for nitrate reductase and xanthine dehydrogenase (CNX) proteins CNX2 and CNX3 in plants (Hoff et al, 1995) and the molybdenum cofactor synthesis (MOCS) proteins MOCS1A and MOCS1B in humans (Haenzelmann et al, 2002). CNX2 and MOCS1A belong to the superfamily of S-adenosylmethionine-dependent radical enzymes, members of which catalyze the formation of protein and/or substrate radicals by reductive cleavage of S-adenosylmethionine by a protein-bound [4Fe-4S] cluster.…”

Section: Introductionmentioning

confidence: 99%

“…While the enzymes of steps 2 to 4 (Matthies et al, 2004;Lardi-Studler et al, 2007;Gehl et al, 2009) have been found to be located in the cytosol, the subcellular localization of step 1 is unclear. Hoff et al (1995) realized that the enzymes involved in step 1, CNX2 and CNX3, carry NH 2 -terminal extensions that may represent targeting signals for the import into chloroplasts or mitochondria. Yet, studies with the in vitro translated proteins and purified chloroplasts and mitochondria failed to show import of these proteins into either of these organelles (Hoff et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

A Novel Role for Arabidopsis Mitochondrial ABC Transporter ATM3 in Molybdenum Cofactor Biosynthesis

et al. 2010

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The molybdenum cofactor (Moco) is a prosthetic group required by a number of enzymes, such as nitrate reductase, sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase. Its biosynthesis in eukaryotes can be divided into four steps, of which the last three are proposed to occur in the cytosol. Here, we report that the mitochondrial ABC transporter ATM3, previously implicated in the maturation of extramitochondrial iron-sulfur proteins, has a crucial role also in Moco biosynthesis. In ATM3 insertion mutants of Arabidopsis thaliana, the activities of nitrate reductase and sulfite oxidase were decreased to ;50%, whereas the activities of xanthine dehydrogenase and aldehyde oxidase, whose activities also depend on iron-sulfur clusters, were virtually undetectable. Moreover, atm3 mutants accumulated cyclic pyranopterin monophosphate, the first intermediate of Moco biosynthesis, but showed decreased amounts of Moco. Specific antibodies against the Moco biosynthesis proteins CNX2 and CNX3 showed that the first step of Moco biosynthesis is localized in the mitochondrial matrix. Together with the observation that cyclic pyranopterin monophosphate accumulated in purified mitochondria, particularly in atm3 mutants, our data suggest that mitochondria and the ABC transporter ATM3 have a novel role in the biosynthesis of Moco.

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Isolation of Two Arabidopsis cDNAs Involved in Early Steps of Molybdenum Cofactor Biosynthesis by Functional Complementation of Escherichia coli Mutants

Cited by 67 publications

References 35 publications

The Molybdenum Cofactor

The Molybdenum Cofactor

The Aspergillus nidulans cnxABC Locus Is a Single Gene Encoding Two Catalytic Domains Required for Synthesis of Precursor Z, an Intermediate in Molybdenum Cofactor Biosynthesis

A Novel Role for Arabidopsis Mitochondrial ABC Transporter ATM3 in Molybdenum Cofactor Biosynthesis

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