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.
Drug Development Diagnostics & Biotechnology Division, CSIR - Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata, 700032, India.
Sagar
S
Lahiri
Shakri
S
Banerjee
Trina
T
Dutta
Shinjinee
S
Sengupta
Sandip
S
Dey
Rusha
R
Roy
Devlina
D
Sengupta
Krishnananda
K
Chattopadhyay
Anil K
AK
Ghosh
D000595:Amino Acid Sequence; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D002614:Chelating Agents / Q000494:pharmacology; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018869:Heat-Shock Response; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D014199:Trehalose / Q000096:biosynthesis*
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D011994:Recombinant Proteins; D014199:Trehalose; C023178:trehalose-phosphatase; D010744:Phosphoric Monoester Hydrolases