Crystal Structures of Cobalamin-independent Methionine Synthase Complexed with Zinc, Homocysteine, and Methyltetrahydrofolate

Ferrer, Jean‐Luc; Ravanel, Stéphane; Robert, Mylène; Dumas, Renaud

doi:10.1074/jbc.c400325200

Cited by 53 publications

(54 citation statements)

References 28 publications

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“…One of the most abundant proteins in the purified sample of thiolated proteins was AtMetS, as shown by its intense spot on 2D-PAGE and by the identification of numerous degradation products in the in vitro labeling study. The recent crystal structure of AtMetS (Ferrer et al, 2004) has identified Cys-649 and Cys-733 as residing in the active site and coordinating a zinc ion. These Cys are therefore likely candidates for thiolation, with this modification likely to inhibit enzyme activity as has been demonstrated with the enzyme in E. coli (Hondorp and Matthews, 2004).…”

Section: Discussionmentioning

confidence: 99%

Stress-Induced Protein S-Glutathionylation in Arabidopsis

et al. 2005

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S-Glutathionylation (thiolation) is a ubiquitous redox-sensitive and reversible modification of protein cysteinyl residues that can directly regulate their activity. While well established in animals, little is known about the formation and function of these mixed disulfides in plants. After labeling the intracellular glutathione pool with [ 35 S]cysteine, suspension cultures of Arabidopsis (Arabidopsis thaliana ecotype Columbia) were shown to undergo a large increase in protein thiolation following treatment with the oxidant tert-butylhydroperoxide. To identify proteins undergoing thiolation, a combination of in vivo and in vitro labeling methods utilizing biotinylated, oxidized glutathione (GSSG-biotin) was developed to isolate Arabidopsis proteins/protein complexes that can be reversibly glutathionylated. Following two-dimensional polyacrylamide gel electrophoresis and matrixassisted laser desorption/ionization time of flight mass spectrometry proteomics, a total of 79 polypeptides were identified, representing a mixture of proteins that underwent direct thiolation as well as proteins complexed with thiolated polypeptides. The mechanism of thiolation of five proteins, dehydroascorbate reductase (AtDHAR1), zeta-class glutathione transferase (AtGSTZ1), nitrilase (AtNit1), alcohol dehydrogenase (AtADH1), and methionine synthase (AtMetS), was studied using the respective purified recombinant proteins. AtDHAR1, AtGSTZ1, and to a lesser degree AtNit1 underwent spontaneous thiolation with GSSG-biotin through modification of active-site cysteines. The thiolation of AtADH1 and AtMetS required the presence of unidentified Arabidopsis proteins, with this activity being inhibited by S-modifying agents. The potential role of thiolation in regulating metabolism in Arabidopsis is discussed and compared with other known redox regulatory systems operating in plants.The tripeptide glutathione (GSH; g-Glu-Cys-Gly) serves important functions in plants as a reductant, transiently accumulating under stress conditions as its oxidized disulfide (GSSG). As well as forming disulfides with itself, GSH can also form mixed disulfides with proteinaceous Cys. This S-glutathionylation of proteins is commonly termed thiolation and has recently become established as a widespread reversible posttranslational modification of proteins that occurs in animal and fungal cells exposed to oxidative stress (Klatt and Lamas, 2000). Although originally considered to be a protective mechanism to prevent the irreversible oxidation of reactive protein-bound Cys to their sulfinic and sulfonic acid derivatives, it is now recognized that thiolation can act as a redox-driven regulator of signal transduction cascades and metabolic pathways (Fratelli et al., 2004). As such, thiolation has clear parallels with redox regulation of proteins mediated through intramolecular or intermolecular disulfide formation with other proteinaceous Cys (Lee et al., 2004). In plants, proteomic approaches have focused upon the redox regulation of proteins forming protein disulfides...

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Section: Discussionmentioning

confidence: 99%

Stress-Induced Protein S-Glutathionylation in Arabidopsis

et al. 2005

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“…4A together with the available structures of MetE (21,53), provides the basis for constructing a homology model of EaCoMT (Fig. 4B) and rationalizing the zinc binding site in the X. autotrophicus enzyme (Fig.…”

Section: Vol 72 2008 Role Of Coenzyme M In Alkene Metabolism 449mentioning

confidence: 99%

Getting a Handle on the Role of Coenzyme M in Alkene Metabolism

Krishnakumar

Sliwa

Endrizzi

et al. 2008

Microbiol Mol Biol Rev

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SUMMARY Coenzyme M (2-mercaptoethanesulfonate; CoM) is one of several atypical cofactors discovered in methanogenic archaea which participate in the biological reduction of CO2 to methane. Elegantly simple, CoM, so named for its role as a methyl carrier in all methanogenic archaea, is the smallest known organic cofactor. It was thought that this cofactor was used exclusively in methanogenesis until it was recently discovered that CoM is a key cofactor in the pathway of propylene metabolism in the gram-negative soil microorganism Xanthobacter autotrophicus Py2. A four-step pathway requiring CoM converts propylene and CO2 to acetoacetate, which feeds into central metabolism. In this process, CoM is used to activate and convert highly electrophilic epoxypropane, formed from propylene epoxidation, into a nucleophilic species that undergoes carboxylation. The unique properties of CoM provide a chemical handle for orienting compounds for site-specific redox chemistry and stereospecific catalysis. The three-dimensional structures of several of the enzymes in the pathway of propylene metabolism in defined states have been determined, providing significant insights into both the enzyme mechanisms and the role of CoM in this pathway. These studies provide the structural basis for understanding the efficacy of CoM as a handle to direct organic substrate transformations at the active sites of enzymes.

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“…The cobalamin-independent enzymes have a molecular mass around 86 kDa; X-ray structures of the enzymes from Arabidopsis thaliana [7] and from the thermophilic bacteria Thermotoga maritima [8] have recently been reported. Steady state kinetic analyses of the cobalamin-independent enzymes from Saccharomyces cerevisiae and Candida albicans, and detailed mechanistic studies on the E. coli metE enzyme, have recently been reported (Suliman et al, 2005;Taurog et al, 2006;Taurog & Matthews, 2006).…”

Section: Introductionmentioning

confidence: 99%

The gene for cobalamin-independent methionine synthase is essential in Candida albicans: A potential antifungal target

Suliman

Appling

Robertus

2007

Archives of Biochemistry and Biophysics

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Methionine synthase catalyzes the transfer of a methyl group from tetrahydrofolate to homocysteine to produce methionine. Although mammalian enzymes are cobalamin-dependent, fungal methionine synthases are cobalamin-independent. The opportunistic pathogen Candida albicans is a diploid and carries two copies of the methionine synthase gene, MET6. Homologous recombination was used to disrupt a single MET6 gene. MET6/met6 knock-outs, deleted with either the URA3 or ARG4 cassette, grew as well as the wild-type strain. However, we were unable to obtain a viable met6/met6 deletion strain, even on media supplemented with exogenous methionine. This suggests that methionine synthase is essential to C. albicans. To explore this further, a C. albicans strain was constructed in which one MET6 locus was deleted and the second placed under a regulatable promoter. The conditional mutant grew well under inducing conditions, even in the absence of methionine. It would not grow under repressing conditions in the absence of methionine, but would grow when the media was supplemented with exogenous methionine. A western blot showed that a small amount of enzyme was expressed under repressing conditions. Taken together, these data reveal that methionine is necessary for growth of C. albicans, but not sufficient -a minimal level of methionine synthase expression is required, perhaps to limit homocysteine toxicity. Furthermore, these results suggest that cobalamin-independent methionine synthase is a plausible target for the design of antifungal agents.

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Crystal Structures of Cobalamin-independent Methionine Synthase Complexed with Zinc, Homocysteine, and Methyltetrahydrofolate

Cited by 53 publications

References 28 publications

Stress-Induced Protein S-Glutathionylation in Arabidopsis

Stress-Induced Protein S-Glutathionylation in Arabidopsis

Getting a Handle on the Role of Coenzyme M in Alkene Metabolism

The gene for cobalamin-independent methionine synthase is essential in Candida albicans: A potential antifungal target

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