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Enzymatic and Regulatory Attributes of Trehalose-6- Phosphate Phosphatase from Candida utilis and its Role During Thermal Stress

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Title Enzymatic and Regulatory Attributes of Trehalose-6-
Phosphate Phosphatase from Candida utilis and its Role During Thermal Stress
 
Creator Lahiri, Sagar
Banerjee, Shakri
Dutta, Trina
Sengupta, Shinjinee
Dey, Sandip
Roy, Rusha
Sengupta, Devlina
Chattopadhyay, Krishnananda
Ghosh, Anil K
 
Subject Drug Development/Diagnostics & Biotechnology
Structural Biology & Bioinformatics
 
Description 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 steadystate
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. Methylationmediated 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 trehalosesynthesis and survivability of the organism under thermal stress was established.
 
Publisher John Wiley & Sons
 
Date 2014
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/2152/1/JOURNAL_OF_CELLULAR_PHYSIOLOGY__V_229__(_9)_1245%2D1255_;2014[56].pdf
Lahiri, Sagar and Banerjee, Shakri and Dutta, Trina and Sengupta, Shinjinee and Dey, Sandip and Roy, Rusha and Sengupta, Devlina and Chattopadhyay, Krishnananda and Ghosh, Anil K (2014) Enzymatic and Regulatory Attributes of Trehalose-6- Phosphate Phosphatase from Candida utilis and its Role During Thermal Stress. Journal of Cellular Physiology, 229 (9). pp. 1245-1255. ISSN 0021-9541
 
Relation http://dx.doi.org/10.1002/jcp.24562
http://www.eprints.iicb.res.in/2152/