scholarly journals Chlamydomonas reinhardtii CNX1E Reconstitutes Molybdenum Cofactor Biosynthesis in Escherichia coli Mutants

2007 ◽  
Vol 6 (6) ◽  
pp. 1063-1067 ◽  
Author(s):  
Ángel Llamas ◽  
Manuel Tejada-Jimenez ◽  
David González-Ballester ◽  
José Javier Higuera ◽  
Guenter Schwarz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Chlamydomonas Reinhardtii ◽  
Double Mutant ◽  
Molybdenum Cofactor ◽  
Wild Type ◽  
Eukaryotic Protein ◽  
E Coli ◽  
Wild Type Phenotype ◽  
Cofactor Biosynthesis ◽  
Molybdenum Cofactor Biosynthesis

ABSTRACT We have isolated and characterized the Chlamydomonas reinhardtii genes for molybdenum cofactor biosynthesis, namely, CNX1G and CNX1E, and expressed them and their chimeric fusions in Chlamydomonas and Escherichia coli. In all cases, the wild-type phenotype was restored in individual mutants as well as in a CNX1G CNX1E double mutant. Therefore, CrCNX1E is the first eukaryotic protein able to complement an E. coli moeA mutant.

scholarly journals Crystal Structure of the Molybdenum Cofactor Biosynthesis Protein MobA from Escherichia coli at Near-Atomic Resolution

Structure ◽  
2000 ◽  
Vol 8 (11) ◽  
pp. 1115-1125 ◽  
Author(s):  
Clare E.M. Stevenson ◽  
Frank Sargent ◽  
Grant Buchanan ◽  
Tracy Palmer ◽  
David M. Lawson
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Molybdenum Cofactor ◽  
Atomic Resolution ◽  
Cofactor Biosynthesis ◽  
Molybdenum Cofactor Biosynthesis

Differential effects of a molybdopterin synthase sulfurylase (moeB) mutation on Escherichia coli molybdoenzyme maturation

2002 ◽  
Vol 80 (4) ◽  
pp. 435-443 ◽  
Author(s):  
Damaraju Sambasivarao ◽  
Raymond J Turner ◽  
Peter T Bilous ◽  
Richard A Rothery ◽  
Gillian Shaw ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Mutant Strain ◽  
Physiological Function ◽  
Signal Sequence ◽  
Molybdenum Cofactor ◽  
Wild Type ◽  
Dmso Reductase ◽  
E Coli ◽  
Membrane Bound

We have generated a chromosomal mutant of moeB (moeBA228T) that demonstrates limited molybdenum cofactor (molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD)) availability in Escherichia coli and have characterized its effect on the maturation and physiological function of two well-characterized respiratory molybdoenzymes: the membrane-bound dimethylsulfoxide (DMSO) reductase (DmsABC) and the membrane-bound nitrate reductase A (NarGHI). In the moeBA228T mutant strain, E. coli F36, anaerobic respiratory growth is possible on nitrate but not on DMSO, indicating that cofactor insertion occurs into NarGHI but not into DmsABC. Fluorescence analyses of cofactor availability indicate little detectable cofactor in the moeBA228T mutant compared with the wild-type, suggesting that NarGHI is able to scavenge limiting cofactor, whereas DmsABC is not. MoeB functions to sulfurylate MoaD, and in the structure of the MoeB–MoaD complex, Ala-228 is located in the interface region between the two proteins. This suggests that the moeBA228T mutation disrupts the interaction between MoeB and MoaD. In the case of DmsABC, despite the absence of cofactor, the twin-arginine signal sequence of DmsA is cleaved in the moeBA228T mutant, indicating that maturation of the holoenzyme is not cofactor-insertion dependent.Key words: mdybdenum cofactor, DMSO reductase, nitrate reductase.

scholarly journals Activity of the Molybdopterin-Containing Xanthine Dehydrogenase of Rhodobacter capsulatus Can Be Restored by High Molybdenum Concentrations in a moeA Mutant Defective in Molybdenum Cofactor Biosynthesis

1999 ◽  
Vol 181 (19) ◽  
pp. 5930-5939 ◽  
Author(s):  
Silke Leimkühler ◽  
Sieglinde Angermüller ◽  
Günter Schwarz ◽  
Ralf R. Mendel ◽  
Werner Klipp
Keyword(s):  
Rhodobacter Capsulatus ◽  
Reductase Activity ◽  
Xanthine Dehydrogenase ◽  
Molybdenum Cofactor ◽  
Transcriptional Unit ◽  
Sequence Alignments ◽  
E Coli ◽  
Cofactor Biosynthesis ◽  
Dimethyl Sulfoxide Reductase ◽  
Molybdenum Cofactor Biosynthesis

ABSTRACT During the screening for Rhodobacter capsulatus mutants defective in xanthine degradation, one Tn5 mutant which was able to grow with xanthine as a sole nitrogen source only in the presence of high molybdate concentrations (1 mM), a phenotype resembling Escherichia coli mogA mutants, was identified. Unexpectedly, the corresponding Tn5 insertion was located within the moeA gene. Partial DNA sequence analysis and interposon mutagenesis of regions flanking R. capsulatus moeA revealed that no further genes essential for molybdopterin biosynthesis are located in the vicinity of moeA and revealed that moeA forms a monocistronic transcriptional unit in R. capsulatus. Amino acid sequence alignments ofR. capsulatus MoeA (414 amino acids [aa]) with E. coli MogA (195 aa) showed that MoeA contains an internal domain homologous to MogA, suggesting similar functions of these proteins in the biosynthesis of the molybdenum cofactor. Interposon mutants defective in moeA did not exhibit dimethyl sulfoxide reductase or nitrate reductase activity, which both require the molybdopterin guanine dinucleotide (MGD) cofactor, even after addition of 1 mM molybdate to the medium. In contrast, the activity of xanthine dehydrogenase, which binds the molybdopterin (MPT) cofactor, was restored to wild-type levels after the addition of 1 mM molybdate to the growth medium. Analysis of fluorescent derivatives of the molybdenum cofactor of purified xanthine dehydrogenase isolated frommoeA and modA mutant strains, respectively, revealed that MPT is inserted into the enzyme only after molybdenum chelation, and both metal chelation and Mo-MPT insertion can occur only under high molybdate concentrations in the absence of MoeA. These data support a model for the biosynthesis of the molybdenum cofactor in which the biosynthesis of MPT and MGD are split at a stage when the molybdenum atom is added to MPT.

scholarly journals Hypersensitivity of Escherichia coli Δ(uvrB-bio) Mutants to 6-Hydroxylaminopurine and Other Base Analogs Is Due to a Defect in Molybdenum Cofactor Biosynthesis

2000 ◽  
Vol 182 (12) ◽  
pp. 3361-3367 ◽  
Author(s):  
Stanislav G. Kozmin ◽  
Youri I. Pavlov ◽  
Ronnie L. Dunn ◽  
Roel M. Schaaper
Keyword(s):  
Escherichia Coli ◽  
Point Mutation ◽  
Excision Repair ◽  
Molybdenum Cofactor ◽  
Transposon Insertion ◽  
Serovar Typhimurium ◽  
Cofactor Biosynthesis ◽  
Molybdenum Cofactor Biosynthesis ◽  
Repair Defect ◽  
Related Compounds

ABSTRACT We have shown previously that Escherichia coli andSalmonella enterica serovar Typhimurium strains carrying a deletion of the uvrB-bio region are hypersensitive to the mutagenic and toxic action of 6-hydroxylaminopurine (HAP) and related base analogs. This sensitivity is not due to theuvrB excision repair defect associated with this deletion because a uvrB point mutation or a uvrAdeficiency does not cause hypersensitivity. In the present work, we have investigated which gene(s) within the deleted region may be responsible for this effect. Using independent approaches, we isolated both a point mutation and a transposon insertion in themoeA gene, which is located in the region covered by the deletion, that conferred HAP sensitivity equal to that conferred by theuvrB-bio deletion. The moeAB operon provides one of a large number of genes responsible for biosynthesis of the molybdenum cofactor. Defects in other genes in the same pathway, such as moa or mod, also lead to the same HAP-hypersensitive phenotype. We propose that the molybdenum cofactor is required as a cofactor for an as yet unidentified enzyme (or enzymes) that acts to inactivate HAP and other related compounds.

scholarly journals Mutations Altering the Predicted Secondary Structure of a Chloroplast 5′ Untranslated Region Affect Its Physical and Biochemical Properties as Well as Its Ability To Promote Translation of Reporter mRNAs Both in the Chlamydomonas reinhardtii Chloroplast and in Escherichia coli

1999 ◽  
Vol 19 (10) ◽  
pp. 6980-6990 ◽  
Author(s):  
David C. Fargo ◽  
John E. Boynton ◽  
Nicholas W. Gillham
Keyword(s):  
Escherichia Coli ◽  
Chlamydomonas Reinhardtii ◽  
Untranslated Region ◽  
Wild Type ◽  
Reporter Expression ◽  
Variable Effect ◽  
E Coli ◽  
Folding Pattern

ABSTRACT Random mutations were generated in the sequence for the 5′ untranslated region (5′UTR) of the Chlamydomonas reinhardtii chloroplast rps7 mRNA by PCR, the coding sequence for the mutant leaders fused upstream of the lacZ′ reporter in pUC18, and transformed into Escherichia coli, and white colonies were selected. Twelve single base pair changes were found at different positions in the rps7 5′UTR in 207 white colonies examined. Seven of the 12 mutant leaders allowed accumulation of abundant lacZ′ message. These mutant rps7leaders were ligated into an aadA expression cassette and transformed into the chloroplast of C. reinhardtii and intoE. coli. In vivo spectinomycin-resistant growth rates and in vitro aminoglycoside adenyltransferase enzyme activity varied considerably between different mutants but were remarkably similar for a given mutant expressed in the Chlamydomonas chloroplast and in E. coli. The variable effect of the mutants onaadA reporter expression and their complete abolition oflacZ′ reporter expression in E. coli suggests differences in the interaction between the 5′UTR of rps7and aadA or lacZ′ coding regions. Severalrps7 5′UTR mutations affected the predicted folding pattern of the 5′UTR by weakening the stability of stem structures. Site-directed secondary mutations generated to restore these structures in the second stem suppressed the loss of reporter activity caused by the original mutations. Additional site-directed mutations that were predicted to further strengthen (A-U→G-C) or weaken (G-C→A-U) the second stem of the rps7 leader both resulted in reduced reporter expression. This genetic evidence combined with differences between mutant and wild-type UV melting profiles and RNase T1 protection gel shifts further indicate that the predicted wild-type folding pattern in the 5′UTR is likely to play an essential role in translation initiation.

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