Trina Dutta

Indian Institute of Chemical Biology, Kolkata, Bengal, India

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Publications (9)25.1 Total impact

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    ABSTRACT: Trehalose-6-phosphate phosphatase (TPP) catalyzes the final step in the biosynthesis of the anti-stress sugar trehalose. An 82 kDa TPP enzyme was isolated from Candida utilis with 61% yield and 43-fold purification. The protein sequence, determined by N-terminal sequencing and MALDI-TOF analysis, showed significant homology with known TPP sequences from related organisms. The full length gene sequence of TPP of Candida utilis was identified using rapid amplification of cDNA ends - PCR reaction (RACE-PCR). The gene was cloned and expressed in E. coli BL21. Recombinant TPP enzyme was isolated using affinity chromatography. CD spectroscopy and steady state fluorescence revealed that the structural and conformational aspects were identical in both native and recombinant forms. The biochemical properties of the two forms were also similar. Km was determined to be ∼0.8 mM. Optimum temperature and pH was found to be 30 °C and 8.5 respectively. Activity was dependent on the presence of divalent cations and inhibited by metal chelators. Methylation mediated regulation of TPP enzyme and its effect on the overall survival of the organism under stress were investigated. The results indicated that enhancement of TPP activity by methylation at the Cysteine residues increased resistance of Candida cells against thermal stress. This work involves extensive investigations towards understanding the physico-chemical properties of the first TPP enzyme from any yeast strain. The mechanism by which methylation regulates its activity has also been studied. A correlation between regulation of trehalose synthesis and survivability of the organism under thermal stress was established. J. Cell. Physiol. 9999: XX-XX, 2014. © 2014 Wiley Periodicals, Inc.
    Journal of Cellular Physiology 01/2014; · 4.22 Impact Factor
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    ABSTRACT: In Saccharomyces cerevisiae methylation at cysteine residue displayed enhanced activity of Trehalose-6-phosphate synthase (TPS). Methods: The cysteine methyltransferase (CMT) responsible for methylating TPS was purified and characterized. The amino acid sequence of the enzyme protein was determined by a combination of N-terminal sequencing and MALDI-TOF/TOF analysis. The nucleotide sequence of the CMT gene was determined, isolated from S. cerevisiae and expressed in E. coli. Targeted disruption of the CMT gene by PCR based homologous recombination in S. cerevisiae was followed by metabolite characterization in the mutant. The purified enzyme was observed to enhance the activity of TPS by a factor of 1.76. The 14kDa enzyme was found to be cysteine specific. The optimum temperature and pH of enzyme activity was calculated as 30°C and 7.0 respectively. The Km Vmax and Kcat against S-adenosyl-L-methionine (AdoMet) were 4.95μM, 3.2U/mg and 6.4s(-1) respectively. Competitive inhibitor S-Adenosyl-L-homocysteine achieved a Ki as 10.9μM against AdoMet. The protein sequence contained three putative AdoMet binding motifs. The purified recombinant CMT activity exhibited similar physicochemical characteristics with the native counterpart. The mutant, Mataα, cmt:: kan(r) exhibited almost 50% reduction in intracellular trehalose concentration. A novel cysteine methyltransferase is purified, which is responsible for enhanced levels of trehalose in S. cerevisiae. General Significance This is the first report about a cysteine methyltransferase which performs S methylation at Cysteine residue regulating TPS activity by 50%, which resulted in an increase of the intercellular stress sugar, trehalose.
    Biochimica et Biophysica Acta 01/2014; · 4.66 Impact Factor
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    ABSTRACT: The present study explored both spontaneous and stress-induced deamidation in acid trehalase and endo-xylanase. An alteration in optimum pH by 1.5 units and optimum temperature by 20 °C accelerated the process of deamidation with a rise in isoaspartate formation and ammonia loss. Spontaneous deamidation during an enzyme-substrate reaction at physiological conditions resulted in accretion of isoaspartyl residues within the enzymes which gradually impaired their catalytic efficacy. Deamidation appeared to be more pronounced in endo-xylanase owing to its secondary structure conformation and high asparagine content. The active sites, Ala 549 in acid trehalase and His184 and Trp188 in endo-xylanase contributed to the loss of enzyme activity as they were flanking the deamidation-susceptible Asn residues. Protein L-isoaspartyl methyl transferase seemed to have a repairing capability, which enabled the heat-damaged enzymes to regain their partial activity as evident from there rise in K (cat)/K (m). Endo-xylanase could regain 38.1 % of its biological activity while a lesser 17.5 % reactivation was obtained in acid trehalase. A unique protein L-isoaspartyl methyl transferase recognition site, Asn 151 was also identified in acid trehalase. A mass increment of the tryptic peptides of repaired enzyme due to methylation catalyzed by protein L-isoaspartyl methyl transferase substantiated the repair hypothesis.
    Applied biochemistry and biotechnology 10/2012; · 1.94 Impact Factor
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    ABSTRACT: Trehalose metabolism plays a central role in various stress responses in yeasts. Methylation dependant enhancement of trehalose synthesis has been reported from yeast Saccharomyces cerevisiae. In order to establish the role of methylation on trehalose metabolism in yeast, it was further investigated in Candida utilis. Universal methyl group donor, S-adenosyl-l-methionine (AdoMet) and its inhibitor, oxidized adenosine (Adox) were used to study the effect of methylation on trehalose metabolism in C. utilis. Treatment of early stationary phase cells of C. utilis with AdoMet and Adox exhibited increase in both intracellular metabolite levels and activities of the trehalose synthesizing enzymes, trehalose-6-phosphate synthase (TPS) and trehalose phosphate phosphatase (TPP). Among the intracellular metabolites studied, trehalose levels were enhanced in presence of AdoMet which correlated with the increasing levels of trehalose synthesizing enzymes. TPS was purified in presence of AdoMet and Adox, following an established protocol reported from this laboratory. Differences in the mobility of control TPS, methylated TPS, and methylation-inhibited TPS during acidic native gel electrophoresis confirmed the occurrence of induced methylation. MALDI-TOF analysis of trypsin-digested samples of the same further strengthened the presence of methylation in TPS. The data presented in this paper strongly indicate a positive role of methylation on trehalose synthesis which finally leads to enhanced trehalose production during the stationary growth phase of C. utilis.
    Carbohydrate research 09/2012; 361C:175-181. · 2.03 Impact Factor
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    ABSTRACT: Trehalose and sucrose, two important anti-stress non-reducing natural disaccharides, are catabolized by two enzymes, namely trehalase and invertase respectively. In this study, a 175 kDa enzyme protein active against both substrates was purified from wild type Candida utilis and characterized in detail. Substrate specificity assay and activity staining revealed the enzyme to be specific for both sucrose and trehalose. The ratio between trehalase and invertase activity was found to be constant at 1:3.5 throughout the entire study. Almost 40-fold purification and 30% yield for both activities were achieved at the final step of purification. The presence of common enzyme inhibitors, thermal and pH stress had analogous effects on its trehalase and invertase activity. Km values for two activities were similar while Vmax and Kcat also differed by a factor of 3.5. Competition plot for both substrates revealed the two activities to be occurring at the single active site. N-terminal sequencing and MALDI-TOF data analysis revealed higher similarity of the purified protein to previously known neutral trehalases. While earlier workers mentioned independent purification of neutral trehalase or invertase from different sources, the present study reports the purification of a single protein showing dual activity.
    Archives of Biochemistry and Biophysics 03/2012; 522(2):90-9. · 3.37 Impact Factor
  • Journal of Molecular Biochemistry. 01/2011;
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    ABSTRACT: The current study was undertaken to correlate post-translational protein modification by methylation with the functionality of enzymes involved in trehalose metabolism in Saccharomyces cerevisiae. Trehalose is an economically important disaccharide providing protection against various kinds of stresses. It also acts as a source of cellular energy by storing glucose. Methyl group donor S-adenosyl L-methionine (AdoMet) and methylation inhibitor-oxidized adenosine (AdOx) were used for the methylation study. AdoMet delayed initial growth of the cells but the overall growth rate remained same suggesting its interference in G1 phase of the cell cycle. Metabolic-altered enzyme activities of acid trehalase (AT), neutral trehalase (NT), and trehalose-6-phosphate synthase (TPS) were observed when treated with AdOx and AdoMet separately. A positive effect of methylation was observed in TPS, hence, it was purified in three different conditions, using AdoMet, AdOx, and control. Differences in mobility of methylated, methylation-inhibited, and control TPS during acidic native gel electrophoresis confirmed the occurrence of induced methylation. Hydrolysis under alkaline pH conditions revealed that methylation of TPS was different than O-methylation. MALDI-TOF analysis of trypsin-digested samples of purified methylated, methylation-inhibited, and control TPS revealed that an increase of 18 Da mass in methylated peptides suggesting the introduction of methyl ester in TPS. Results of amino acid analysis corroborated the presence of methyl cysteine. The data presented here strongly suggests that trehalose production was enhanced due to methylation of TPS arising from carboxymethylation of cysteine residues.
    Journal of Cellular Physiology 01/2011; 226(1):158-64. · 4.22 Impact Factor
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    ABSTRACT: S-Adenosyl-L-Methionine (AdoMet), S-Adenosyl-L-Homocysteine (AdoHcy), adenosine, trehalose and oleanolic acid were measured in six me-dicinal herbs and three spices. The findings showed that AdoMet content was forty six fold higher in the leaves of Catharanthus roseus as compared with average AdoMet content of rest of the plants. In comparison to other plants, Withania somnifera had very high AdoHcy: Ado-Met and adenosine: AdoMet ratios indicating it may have contained high AdoMet. Trehalose was found to be twenty fold and nine fold higher in bulb of Allium cepa and root of Withania somnifera respectively with respect to average trehalose content of rest of the plants. Ocimum sanctum appeared to be a rich source of olea-nolic acid. It appeared from our study that Ca-tharanthus roseus, Allium cepa and Ocimum sanctum could be utilized as natural sources of AdoMet, trehalose and oleanolic acid respec-tively.
    01/2010; 2:968-972.
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    ABSTRACT: Purified trehalose-6-phosphate synthase (TPS) of Saccharomyces cerevisiae was effective over a wide range of substrates, although differing with regard to their relative activity. Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity, particularly when a pyrimidine glucose nucleotide like UDPG was used, rather than a purine glucose nucleotide like GDPG. A high V(max) and a low K(m) value of UDPG show its greater affinity with TPS than GDPG or TDPG. Among the glucosyl acceptors TPS showed maximum activity with G-6-P which was followed by M-6-P and F-6-P. Effect of heparin was also extended to the purification of TPS activity, as it helped to retain both stability and activity of the final purified enzyme. Metal co-factors, specifically MnCl(2) and ZnCl(2) acted as stimulators, while enzyme inhibitors had very little effect on TPS activity. Metal chelators like CDTA, EGTA stimulated enzyme activity by chelation of metal inhibitors. Temperature and pH optima of the purified enzyme were determined to be 40 degrees C and pH 8.5 respectively. Enzyme activity was stable at 0-40 degrees C and at alkaline pH.
    Biochimica et Biophysica Acta 04/2009; 1790(5):368-74. · 4.66 Impact Factor