Enzymatic attributes of an l-isoaspartyl methyltransferase from Candida utilis and its role in cell survival.

BACKGROUNDS: Spontaneous deamidation and isoaspartate (IsoAsp) formation contributes to aging and reduced longevity in cells. A protein-l-isoaspartate (d-aspartate) O-methyltransferase (PCMT) is responsible for minimizing IsoAsp moieties in most organisms. METHODS: PCMT was purified in its native form from yeast Candida utilis. The role of the native PCMT in cell survival and protein repair was investigated by manipulating intracellular PCMT levels with Oxidized Adenosine (AdOx) and Lithium Chloride (LiCl). Proteomic Identification of possible cellular targets was carried out using 2-dimensional gel electrophoresis, followed by on-Blot methylation and mass spectrometric analysis. RESULTS: The 25.4 kDa native PCMT from C. utilis was found to have a Km of 3.5 µM for AdoMet and 33.36 µM for IsoAsp containing Delta Sleep Inducing Peptide (DSIP) at pH 7.0. Native PCMT comprises of 232 amino acids which is coded by a 698 bp long nucleotide sequence. Phylogenetic comparison revealed the PCMT to be related more closely with the prokaryotic homologs. Increase in PCMT levels in vivo correlated with increased cell survival under physiological stresses. PCMT expression was seen to be linked with increased intracellular reactive oxygen species (ROS) concentration. Proteomic identification of possible cellular substrates revealed that PCMT interacts with proteins mainly involved with cellular housekeeping. PCMT effected both functional and structural repair in aged proteins in vitro. GENERAL SIGNIFICANCE: Identification of PCMT in unicellular eukaryotes like C. utilis promises to make investigations into its control machinery easier owing to the familiarity and flexibility of the system.

Enzymatic and regulatory attributes of trehalose-6-phosphate phosphatase from Candida utilis and its role during thermal stress.

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 C. utilis was identified using rapid amplification of cDNA ends-PCR reaction (RACE-PCR). The gene was cloned and expressed in Escherichia 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 were 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 toward 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.

Purification, characterization, sequencing and molecular cloning of a novel cysteine methyltransferase that regulates trehalose-6-phosphate synthase from Saccharomyces cerevisiae.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Methylation dependent enhancement of trehalose production in Candida utilis.

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.

Arginine mediated purification of trehalose-6-phosphate synthase (TPS) from Candida utilis: Its characterization and regulation.

BACKGROUND: 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). METHODS: 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). RESULTS: 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. GENERAL SIGNIFICANCE: 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.

Possible regulation of trehalose metabolism by methylation in Saccharomyces cerevisiae.

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.