Cloning, expression and characterization of trehalose-6-phosphate phosphatase from a psychrotrophic bacterium, Arthrobacter strain A3.
A trehalose-6-phosphate phosphatase (TPP) gene, otsB, from a psychrotrophic bacterium, Arthrobacter strain A3, was identified. The product of this otsB gene is 266 amino acids in length with a calculated molecular weight of 27,873 Da. The protein was expressed in Escherichia coli and purified to apparent homogeneity. The purified recombinant TPP catalyzed the dephosphorylation of trehalose-6-phosphate to form trehalose and showed a broad optimum pH range from 5.0 to 7.5. This enzyme also showed an absolute requirement for Mg(2+) or Co(2+) for catalytic activity. The recombinant TPP had a maximum activity at 30 °C and maintained activity over a temperature range of 4-30 °C. TPP was generally heat-labile, losing 70 % of its activity when subjected to heat treatment at 50 °C for 6 min. Kinetic analysis of the Arthrobacter strain A3 TPP showed ~tenfold lower K (m) values when compared with values derived from other bacterial TPP enzymes. The highest k (cat)/K (m) value was 37.5 mM(-1) s(-1) (repeated three times), which is much higher than values published for mesophilic E. coli TPP, indicating that the Arthrobacter strain A3 TPP possessed excellent catalytic activity at low temperatures. Accordingly, these characteristics suggest that the TPP from the Arthrobacter strain A3 is a new cold-adapted enzyme. In addition, this is the first report characterizing the enzymatic properties of a TPP from a psychrotrophic organism.
Key Laboratory of Arid and Grassland Agroecology of the Ministry of Education, School of Life Sciences, Lanzhou University, Lanzhou, 730000, People's Republic of China.
Yuan-Ting
YT
Li
Hai-Hong
HH
Zhang
Hong-Mei
HM
Sheng
Li-Zhe
LZ
An
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001482:Base Composition; D003001:Cloning, Molecular; D003080:Cold Temperature; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013403:Sugar Phosphates / Q000378:metabolism; D014199:Trehalose / Q000031:analogs & derivatives / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D013403:Sugar Phosphates; C082722:trehalose-6-phosphate; D014199:Trehalose; C023178:trehalose-phosphatase; D010744:Phosphoric Monoester Hydrolases