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author:("Yokota, agiho")
1.  MtnBD Is a Multifunctional Fusion Enzyme in the Methionine Salvage Pathway of Tetrahymena thermophila 
PLoS ONE  2013;8(7):e67385.
To recycle reduced sulfur to methionine in the methionine salvage pathway (MSP), 5-methylthioribulose-1-phosphate is converted to 2-keto-4-methylthiobutyrate, the methionine precursor, by four steps; dehydratase, enolase, phosphatase, and dioxygenase reactions (catalyzed by MtnB, MtnW, MtnX and MtnD, respectively, in Bacillus subtilis). It has been proposed that the MtnBD fusion enzyme in Tetrahymena thermophila catalyzes four sequential reactions from the dehydratase to dioxygenase steps, based on the results of molecular biological analyses of mutant yeast strains with knocked-out MSP genes, suggesting that new catalytic function can be acquired by fusion of enzymes. This result raises the question of how the MtnBD fusion enzyme can catalyze four very different reactions, especially since there are no homologous domains for enolase and phosphatase (MtnW and MtnX, respectively, in B. subtilis) in the peptide. Here, we tried to identify the domains responsible for catalyzing the four reactions using recombinant proteins of full-length MtnBD and each domain alone. UV-visible and 1H-NMR spectral analyses of reaction products revealed that the MtnB domain catalyzes dehydration and enolization and the MtnD domain catalyzes dioxygenation. Contrary to a previous report, conversion of 5-methylthioribulose-1-phosphate to 2-keto-4-methylthiobutyrate was dependent on addition of an exogenous phosphatase from B. subtilis. This was observed for both the MtnB domain and full-length MtnBD, suggesting that MtnBD does not catalyze the phosphatase reaction. Our results suggest that the MtnB domain of T. thermophila MtnBD acquired the new function to catalyze both the dehydratase and enolase reactions through evolutionary gene mutations, rather than fusion of MSP genes.
doi:10.1371/journal.pone.0067385
PMCID: PMC3698126  PMID: 23840871
2.  Crystallization and preliminary X-ray analysis of 2,3-diketo-5-methylthiopentyl-1-phosphate enolase from Bacillus subtilis  
Crystals of the 45.1 kDa functional form of 2,3-diketo-5-methylthiopentyl-1-phosphate enolase from B. subtilis diffracted to 2.30 Å resolution.
2,3-Diketo-5-methylthiopentyl-1-phosphate enolase (DK-MTP-1P enolase) from Bacillus subtilis was crystallized using the hanging-drop vapour-diffusion method. Crystals grew using PEG 3350 as the precipitant at 293 K. The crystals diffracted to 2.3 Å resolution at 100 K using synchrotron radiation and were found to belong to the monoclinic space group P21, with unit-cell parameters a = 79.3, b = 91.5, c = 107.0 Å, β = 90.8°. The asymmetric unit contained four molecules of DK-MTP-1P enolase, with a V M value of 2.2 Å3 Da−1 and a solvent content of 43%.
doi:10.1107/S174430910804311X
PMCID: PMC2635871  PMID: 19194007
methionine-salvage pathway; Bacillus subtilis; RuBisCO; RuBisCO-like proteins; 2,3-diketo-5-methylthiopentyl-1-phosphate enolase
3.  Crystallization and preliminary X-ray analysis of methylthioribose-1-phosphate isomerase from Bacillus subtilis  
Crystals of the 39 kDa functional form of methylthioribose-1-phosphate isomerase from B. subtilis diffracted to 2.50 Å.
Methylthioribose-1-phosphate isomerase (MtnA) from Bacillus subtilis, the first enzyme in the downstream section of the methionine-salvage pathway, was crystallized using the sitting-drop vapour-diffusion method. Crystals grew using ammonium sulfate as the precipitant at 293 K. They diffracted to 2.5 Å at 100 K using synchrotron radiation and were found to belong to the tetragonal space group P41, with unit-cell parameters a = b = 69.2, c = 154.7 Å. The asymmetric unit contains two molecules of MtnA, with a V M value of 2.4 Å3 Da−1 and a solvent content of 48%.
doi:10.1107/S1744309105015757
PMCID: PMC1952323  PMID: 16511105
methylthioribose-1-phosphate; methylthioribulose-1-phosphate; methylthioadenosine
4.  Bacterial variations on the methionine salvage pathway 
BMC Microbiology  2004;4:9.
Background
The thiomethyl group of S-adenosylmethionine is often recycled as methionine from methylthioadenosine. The corresponding pathway has been unravelled in Bacillus subtilis. However methylthioadenosine is subjected to alternative degradative pathways depending on the organism.
Results
This work uses genome in silico analysis to propose methionine salvage pathways for Klebsiella pneumoniae, Leptospira interrogans, Thermoanaerobacter tengcongensis and Xylella fastidiosa. Experiments performed with mutants of B. subtilis and Pseudomonas aeruginosa substantiate the hypotheses proposed. The enzymes that catalyze the reactions are recruited from a variety of origins. The first, ubiquitous, enzyme of the pathway, MtnA (methylthioribose-1-phosphate isomerase), belongs to a family of proteins related to eukaryotic intiation factor 2B alpha. mtnB codes for a methylthioribulose-1-phosphate dehydratase. Two reactions follow, that of an enolase and that of a phosphatase. While in B. subtilis this is performed by two distinct polypeptides, in the other organisms analyzed here an enolase-phosphatase yields 1,2-dihydroxy-3-keto-5-methylthiopentene. In the presence of dioxygen an aci-reductone dioxygenase yields the immediate precursor of methionine, ketomethylthiobutyrate. Under some conditions this enzyme produces carbon monoxide in B. subtilis, suggesting a route for a new gaseous mediator in bacteria. Ketomethylthiobutyrate is finally transaminated by an aminotransferase that exists usually as a broad specificity enzyme (often able to transaminate aromatic aminoacid keto-acid precursors or histidinol-phosphate).
Conclusion
A functional methionine salvage pathway was experimentally demonstrated, for the first time, in P. aeruginosa. Apparently, methionine salvage pathways are frequent in Bacteria (and in Eukarya), with recruitment of different polypeptides to perform the needed reactions (an ancestor of a translation initiation factor and RuBisCO, as an enolase, in some Firmicutes). Many are highly dependent on the presence of oxygen, suggesting that the ecological niche may play an important role for the existence and/or metabolic steps of the pathway, even in phylogenetically related bacteria. Further work is needed to uncover the corresponding steps when dioxygen is scarce or absent (this is important to explore the presence of the pathway in Archaea). The thermophile T. tengcongensis, that thrives in the absence of oxygen, appears to possess the pathway. It will be an interesting link to uncover the missing reactions in anaerobic environments.
doi:10.1186/1471-2180-4-9
PMCID: PMC395828  PMID: 15102328
5.  Citrulline and DRIP‐1 Protein (ArgE Homologue) in Drought Tolerance of Wild Watermelon 
Annals of Botany  2002;89(7):825-832.
Drought‐affected plants experience more than just desiccation of their organs due to water deficit. Plants transpire 1000 times more molecules of water than of CO2 fixed by photosynthesis in full sunlight. One effect of transpiration is to cool the leaves. Accordingly, drought brings about such multi‐stresses as high temperatures, excess photoradiation and other factors that affect plant viability. Wild watermelon serves as a suitable model system to study drought responses of C3 plants, since this plant survives drought by maintaining its water content without any wilting of leaves or desiccation even under severe drought conditions. Under drought conditions in the presence of strong light, wild watermelon accumulates high concentrations of citrulline, glutamate and arginine in its leaves. The accumulation of citrulline and arginine may be related to the induction of DRIP‐1, a homologue of ArgE in Escherichia coli, where it functions to incorporate the carbon skeleton of glutamate into the urea cycle. Immunogold electron microscopy reveals the enzyme to be confined exclusively to the cytosol. DRIP‐1 is also induced by treating wild watermelon with 150 mm NaCl, but is not induced following treatment with 100 µm abscisic acid. The salt treatment causes the accumulation of γ‐aminobutyrate, glutamine and alanine, in addition to a smaller amount of citrulline. Citrulline may function as a potent hydroxyl radical scavenger.
doi:10.1093/aob/mcf074
PMCID: PMC4233801  PMID: 12102508
Wild watermelon (Citrullus lanatus L.); drought; photosynthesis; electron sink; active oxygen; ascorbate peroxidase; citrulline; arginine; γ‐aminobutyrate, ArgE

Results 1-5 (5)