<i>Chlamydomonas reinhardtii</i>
            CNX1E Reconstitutes Molybdenum Cofactor Biosynthesis in
            <i>Escherichia coli</i>
            Mutants

Llamas, Ángel; Tejada‐Jiménez, Manuel; González-Ballester, David; Higuera, José Javier; Schwarz, Günter; Galván, Aurora; Fernández, Emilio

doi:10.1128/ec.00072-07

Cited by 24 publications

(20 citation statements)

References 15 publications

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“…In order to determine the relationship between N-hydroxylated compound (NHC) toxicity and Moco, we have investigated the phenotypes of different Chlamydomonas Moco mutants in the presence of HAP. Screening the Chlamydomonas mutant library generated in our lab by insertional mutagenesis (7) allowed us to identify five mutations in Moco biosynthesis genes (21). These strains are affected at genes Cnx2 and Cnx3 of the first step of Moco biosynthesis, Cnx5 of the second step, Cnx1G of the third step, and Cnx1E of the fourth step (see introduction).…”

Section: Resultsmentioning

confidence: 99%

“…Moco is widespread in all kingdoms and synthesized by a conserved pathway, divided in several steps according to the biosynthesis of its intermediates from a guanosine derivative (probably GTP): cyclic pyranopterin monophosphate (cPMP), MPT, and MPT-AMP (adenylated molybdopterin). In Chlamydomonas reinhardtii, the CNX2 and CNX3 enzymes catalyze the conversion of GTP into cPMP, CNX5, CNX6, and CNX7 from cPMP into MPT, CNX1G from MPT into MPT-AMP, and CNX1E from MPT-AMP into Moco (21). Two families of Moco-containing enzymes are present in eukaryotes, the sulfite oxidase (SO) family and the xanthine oxidase (XO) family.…”

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confidence: 99%

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The Chlamydomonas reinhardtii Molybdenum Cofactor Enzyme crARC Has a Zn-Dependent Activity and Protein Partners Similar to Those of Its Human Homologue

et al. 2011

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The ARC (amidoxime reducing component) proteins are molybdenum cofactor (Moco) enzymes named hmARC1 and hmARC2 (human ARCs [hmARCs]) in humans and YcbX in Escherichia coli. They catalyze the reduction of a broad range of N-hydroxylated compounds (NHC) using reducing power supplied by other proteins. Some NHC are prodrugs or toxic compounds. YcbX contains a ferredoxin (Fd) domain and requires the NADPH flavin reductase CysJ to reduce NHC. In contrast, hmARCs lack the Fd domain and require a human cytochrome b5 (hCyt b5) and a human NADH Cyt b5 reductase (hCyt b5-R) to reduce NHC. The ARC proteins in the plant kingdom are uncharacterized. We demonstrate that Chlamydomonas reinhardtii mutants defective in Moco biosynthesis genes are sensitive to the NHC N 6 -hydroxylaminopurine (HAP). The Chlamydomonas reinhardtii ARC protein crARC has been purified and characterized. The six Chlamydomonas Fds were isolated, but none of them are required by crARC to reduce HAP. We have also purified and characterized five C. reinhardtii Cyt b5 (crCyt b5) and two flavin reductases, one that is NADPH dependent (crCysJ) and one that is NADH dependent (crCyt b5-R). The data show that crARC uses crCyt b5-1 and crCyt b5-R to reduce HAP. The crARC has a Zn-dependent activity, and the presence of Zn increases its V max more than 14-fold. In addition, all five cysteines of crARC were substituted by alanine, and we demonstrate that the fully conserved cysteine 252 is essential for both Moco binding and catalysis. Therefore, it is proposed that crARC belongs to the sulfite oxidase family of Moco enzymes.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

The Chlamydomonas reinhardtii Molybdenum Cofactor Enzyme crARC Has a Zn-Dependent Activity and Protein Partners Similar to Those of Its Human Homologue

et al. 2011

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“…Third, those mutants overexpressing the ARS marker activity in the presence of nitrate were unable to grow on nitrate, NIT Ϫ . These mutants are affected at NIA1, Moco biosynthesis genes, and new genes currently under study (21,32). Finally, NIT ϩ mutants expressing ARS in ammoniumcontaining medium, proposed to be affected by the negative signaling of the pathway by ammonium or ammonium derivatives, were isolated.…”

Section: Regulatory Genes For Nitrate Assimilationmentioning

confidence: 99%

Nitrate Assimilation inChlamydomonas

2008

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“…The molybdate insertion reaction involves both functional domains of Mo-insertases, namely Eand G-domain [7] whose role for the Mo-insertion reaction has been studied in detail using the plant (Arabidopsis thaliana) Mo-insertase Cnx1 as model enzyme [8][9][10][11][12][13][14][15]. Notably, these domains are reactive as separately expressed domains (prokaryotes) or fused together (eukaryotes, except the lower alga Chlamydomonas reinhardtii [16]). Initially, the Mo-insertase G-domain catalyzes the ligation of an AMP molecule to the terminal phosphate group of MPT, yielding adenylated MPT (MPT-AMP, [11,12]).…”

Section: Introductionmentioning

confidence: 99%

Insights into the Cnx1E catalyzed MPT-AMP hydrolysis

et al. 2020

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Molybdenum insertases (Mo-insertases) catalyze the final step of molybdenum cofactor (Moco) biosynthesis, an evolutionary old and highly conserved multi-step pathway. In the first step of the pathway, GTP serves as substrate for the formation of cyclic pyranopterin monophosphate, which is subsequently converted into molybdopterin (MPT) in the second pathway step. In the following synthesis steps, MPT is adenylated yielding MPT-AMP that is subsequently used as substrate for enzyme catalyzed molybdate insertion. Molybdate insertion and MPT-AMP hydrolysis are catalyzed by the Mo-insertase E-domain. Earlier work reported a highly conserved aspartate residue to be essential for Mo-insertase functionality. In this work, we confirmed the mechanistic relevance of this residue for the Arabidopsis thaliana Mo-insertase Cnx1E. We found that the conservative substitution of Cnx1E residue Asp274 by Glu (D274E) leads to an arrest of MPT-AMP hydrolysis and hence to the accumulation of MPT-AMP. We further showed that the MPT-AMP accumulation goes in hand with the accumulation of molybdate. By crystallization and structure determination of the Cnx1E variant D274E, we identified the potential reason for the missing hydrolysis activity in the disorder of the region spanning amino acids 269 to 274. We reasoned that this is caused by the inability of a glutamate in position 274 to coordinate the octahedral Mg2+-water complex in the Cnx1E active site.

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Chlamydomonas reinhardtii CNX1E Reconstitutes Molybdenum Cofactor Biosynthesis in Escherichia coli Mutants

Cited by 24 publications

References 15 publications

The Chlamydomonas reinhardtii Molybdenum Cofactor Enzyme crARC Has a Zn-Dependent Activity and Protein Partners Similar to Those of Its Human Homologue

The Chlamydomonas reinhardtii Molybdenum Cofactor Enzyme crARC Has a Zn-Dependent Activity and Protein Partners Similar to Those of Its Human Homologue

Nitrate Assimilation inChlamydomonas

Insights into the Cnx1E catalyzed MPT-AMP hydrolysis

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