title: Purification, characterization, sequencing and molecular cloning of a novel cysteine methyltransferase that regulates trehalose-6-phosphate synthase from Saccharomyces cerevisiae.
abstract: 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.
  
  
  CONCLUSIONS
  A novel cysteine methyltransferase is purified, which is responsible for enhanced levels of trehalose in S. cerevisiae.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Drug Development Diagnostics and Biotechnology Division, Indian Institute of Chemical Biology, Jadavpur, Kolkata, West Bengal 700032, India.
authors.forename: Shinjinee
authors.initials: S
authors.lastname: Sengupta
authors.affiliation: Department of Drug Development Diagnostics and Biotechnology Division, Indian Institute of Chemical Biology, Jadavpur, Kolkata, West Bengal 700032, India.
authors.forename: Shakri
authors.initials: S
authors.lastname: Banerjee
authors.affiliation: Department of Drug Development Diagnostics and Biotechnology Division, Indian Institute of Chemical Biology, Jadavpur, Kolkata, West Bengal 700032, India.
authors.forename: Sagar
authors.initials: S
authors.lastname: Lahiri
authors.affiliation: Department of Drug Development Diagnostics and Biotechnology Division, Indian Institute of Chemical Biology, Jadavpur, Kolkata, West Bengal 700032, India.
authors.forename: Trina
authors.initials: T
authors.lastname: Dutta
authors.affiliation: Department of Drug Development Diagnostics and Biotechnology Division, Indian Institute of Chemical Biology, Jadavpur, Kolkata, West Bengal 700032, India.
authors.forename: Tarun K
authors.initials: TK
authors.lastname: Dhar
authors.affiliation: Department of Drug Development Diagnostics and Biotechnology Division, Indian Institute of Chemical Biology, Jadavpur, Kolkata, West Bengal 700032, India. Electronic address: anilghosh51@gmail.com.
authors.forename: Anil K
authors.initials: AK
authors.lastname: Ghosh
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005964:Glucosyltransferases / Q000378:metabolism*; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D014199:Trehalose / Q000378:metabolism
chemical_list: D014199:Trehalose; D008780:Methyltransferases; D005964:Glucosyltransferases; C023180:trehalose-6-phosphate synthase; D003545:Cysteine
keywords: AdoMet; Adox; Methylation; Trehalose