Publications (2)4.7 Total impact
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Article: Enzymatic synthesis of dTDP-4-amino-4,6-dideoxy-D-glucose using GerB (dTDP-4-keto-6-deoxy-D-glucose aminotransferase).
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ABSTRACT: Over-expressed GerB (dTDP-4-keto-6-deoxy-d-glucose aminotransferase) of Streptomyces sp. GERI-155 was used in the enzymatic synthesis of dTDP-4-amino-4,6-dideoxy-D-glucose (2) from dTDP-4-keto-6-deoxy-D-glucose (1). [Carbohydrate structure: see text]. Five enzymes including dTMP kinase (TMK), acetate kinase (ACK), dTDP-glucose synthase (TGS), dTDP-glucose 4,6-dehydratase (DH), and dTDP-4-keto-6-deoxy-d-glucose aminotransferase (GerB) were used to synthesize 2 on a large scale from glucose-1-phosphate and TMP. A conversion yield of up to 57% was obtained by HPLC peak integration given a reaction time of 270min. After purification by two successive preparative HPLC systems, the final product was identified by HPLC and then analyzed by (1)H, (13)C, (1)H-(1)H COSY NMR spectrometry.Carbohydrate Research 09/2007; 342(11):1412-8. · 2.33 Impact Factor -
Article: Biochemical characterization of a novel thermostable glucose-1-phosphate thymidylyltransferase from Thermuscaldophilus: Probing the molecular basis for its unusual thermostability
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ABSTRACT: 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: Mg2+ > Zn2+ > Cu2+ > Co2+ > Fe2+ > Ca2+ > Fe3+>Ni2+. 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.Enzyme and Microbial Technology 37(4):402-409. · 2.37 Impact Factor
