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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. · 2.93 Impact Factor
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ABSTRACT: Trehalose is the most important multifunctional, non-reducing disaccharide found in nature. It is synthesized in yeast by an enzyme complex: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP).
In the present study TPS is purified using a new methodology from Candida utilis cells by inclusion of 100mM l-arginine during cell lysis and in the mobile phase of high performance gel filtration liquid chromatography (HPGFLC).
An electrophoretically homogenous TPS that was purified was a 60 kDa protein with 22.1 fold purification having a specific activity of 2.03 U/mg. Alignment of the N-terminal sequence with TPS from Saccharomyces cerevisiae confirmed the 60 kDa protein to be TPS. Optimum activity of TPS was observed at a protein concentration of 1 μg, at a temperature of 37°C and pH 8.5. Aggregation mediated enzyme regulation was indicated. Metal cofactors, especially MnCl₂, MgCl₂ and ZnSO₄, acted as stimulators. Metal chelators like CDTA and EGTA stimulated enzyme activity. Among the four glucosyl donors, the highest V(max) and lowest K(m) values were calculated as 2.96 U/mg and 1.36 mM when adenosine di phosphate synthase (ADPG) was used as substrate. Among the glucosyl acceptors, glucose-6-phosphate (G-6-P) showed maximum activity followed by fructose-6-phosphate (F-6-P). Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity with different glucosyl donors.
Substrate specificity, V(max) and K(m) values provided an insight into an altered trehalose metabolic pathway in the C. utilis strain where ADPG is the preferred substrate rather than the usual substrate uridine diphosphaphate glucose (UDPG). The present work employs a new purification strategy as well as highlights an altered pathway in C. utilis.
Biochimica et Biophysica Acta 07/2011; 1810(12):1346-54. · 4.66 Impact Factor
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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. · 3.87 Impact Factor
