Aspartate aminotransferase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC 125

• Published in: European Journal of Biochemistry
• Main Authors: Birolo, Leila, Tutino, M. Luisa, Fontanella, Bianca, Gerday, Charles, Mainolfi, Katia, Pascarella, Stefano, Sannia, Giovanni, Vinci, Floriana, Marino, Gennaro
• Format: Article in Journal/Newspaper
• Language: English
• Published: Wiley 2000
• Subjects: Antarc*; Antarctic
• Online Access: http://dx.doi.org/10.1046/j.1432-1327.2000.01299.x; https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1046%2Fj.1432-1327.2000.01299.x; https://febs.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1432-1327.2000.01299.x

The gene encoding aspartate aminotransferase from the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 was cloned, sequenced and overexpressed in Escherichia coli . The recombinant protein ( Ph AspAT) was characterized both at the structural and functional level in comparison with the E. coli enzyme ( Ec AspAT), which is the most closely related (52% sequence identity) bacterial counterpart. Ph AspAT is rapidly inactivated at 50 °C (half‐life = 6.8 min), whereas at this temperature Ec AspAT is stable for at least 3 h. The optimal temperature for Ph AspAT activity is ≈ 64 °C, which is some 11 °C below that of Ec AspAT. The protein thermal stability was investigated by following changes in both tryptophan fluorescence and amide ellipticity; this clearly suggested that a first structural transition occurs at ≈ 50 °C for Ph AspAT. These results agree with the expected thermolability of a psychrophilic enzyme, although the observed stability is much higher than generally found for enzymes isolated from cold‐loving organisms. Furthermore, in contrast with the higher efficiency exhibited by several extracellular psychrophilic enzymes, both k cat and k cat / K m of Ph AspAT are significantly lower than those of Ec AspAT over the whole temperature range. This behaviour possibly suggests that the adaptation of this class of endocellular enzymes to a cold environment may have only made them less stable and not more efficient. The affinity of Ph AspAT for both amino‐acid and 2‐oxo‐acid substrates decreases with increasing temperature. However, binding of maleate and 2‐methyl‐ l ‐aspartate, which both inhibit the initial steps of catalysis, does not change over the temperature range tested. Therefore, the observed temperature effect may occur at any of the steps of the catalytic mechanism after the formation of the external aldimine. A molecular model of Ph AspAT was constructed on the basis of sequence homology with other AspATs. Interestingly, it shows no insertion or extension of loops, but some cavities and ...