Biochemical characterization of a novel thermostable glucose-1-phosphate thymidylyltransferase from Thermuscaldophilus: Probing the molecular basis for its unusual thermostability

• Published in: Enzyme and microbial technology 2005-09, Vol.37 (4), p.402-409
• Main Authors: Parajuli, Niranjan, Basnet, Devi B., Chung, Young Soo, Lee, Hei Chan, Liou, Kwangkyoung, Sohng, Jae Kyung
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Codon usage; dTTP; Fundamental and applied biological sciences. Psychology; Glucose-1-phosphate thymidylyltransferase; Miscellaneous; Mission oriented research; Thermostability; Thermus caldophilus GK24; β-Sheets
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2005.02.024

We have found that the thermophilic activity of glucose-1-phosphate thymidylyltransferase (stRmlA) is conditional upon the presence certain substances. In particular, it showed its thermal stability only in the presence of 50 mM dTTP or dTMP, thermal stability being the highest at 70 °C. The purified enzyme was stable up to 90 °C within a broad pH range from 2.0 to 13.0, and its maximum activity was measured at a pH of 11.5 at 70 °C. Unlike other mesophilic counterparts, it showed catalytic activity in the presence of various metal ions in the following order of reactivity: Mg 2+ > Zn 2+ > Cu 2+ > Co 2+ > Fe 2+ > Ca 2+ > Fe 3+>Ni 2+. Its catalytic activity was not inhibited even by the denaturants 50 mM guanidine hydrochloride and 50 mM urea. To explore the molecular basis for its unusual thermostability, homology structural modeling, codon usage comparisons, and amino acid composition analyses were performed. The CCC, CUC and UCC codons are significantly higher, and distributed uniformly in strmlA as contrasted with other GPTTs. Our analyses revealed that the GC rich coding sequences may not be the reason for thermostability of stRmlA. The β-sheets are also found more likely in stRmlA, which contributes to thermal stability by increasing the number of hydrogen bonds. Further results suggest that a large number of apolar functional groups exposed to solvent accessible surface area, a significant number of residues sensitive to oxidation or deamination, and a higher hydrophobicity, any or all of the which could be reasons for its unique thermophilic behavior. Finally, we postulate that dTTP or dTMP enters the active site and binds tightly to inhibit the defolding of the protein, which, in turn, increases its thermal stability. Such findings will be useful for further investigations on thermophilic behavior of enzymes.