Aspartate aminotransferase from the Antarctic bacterium - Pseudoalteromonas haloplanktis TAC 125 - Cloning, expression, properties, and molecular modelling

The gene encoding aspartate aminotransferase from the psychrophilic bacteri um Pseudoalteromonas haloplanktis TAC 125 was cloned, sequenced and overexp ressed in Escherichia coli. The recombinant protein (PhAspAT) was character ized both at the structural and functional level in comparison with the E. coli enzyme (EcAspAT), which is the most closely related (52% sequence iden tity) bacterial counterpart. PhAspAT is rapidly inactivated at 50 degrees C (half-life = 6.8 min), whereas at this temperature EcAspAT is stable for a t least 3 h. The optimal temperature for PhAspAT activity is approximate to 64 degrees C, which is some 11 degrees C below that of EcAspAT. The protei n thermal stability was investigated by following changes in both tryptopha n fluorescence and amide ellipticity; this clearly suggested that a first s tructural transition occurs at approximate to 50 degrees C for PhAspAT. The se results agree with the expected thermolability of a psychrophilic enzyme , although the observed stability is much higher than generally found for e nzymes isolated from cold-loving organisms. Furthermore, in contrast with t he higher efficiency exhibited by several extracellular psychrophilic enzym es, both k(cat) and k(cat)/K-m of PhAspAT are significantly lower than thos e of EcAspAT over the whole temperature range. This behaviour possibly sugg ests that the adaptation of this class of endocellular enzymes to a cold en vironment may have only made them less stable and not more efficient. The affinity of PhAspAT for both amino-acid and 2-oxo-acid substrates decre ases with increasing temperature. However, binding of maleate and 2-methyl- L-aspartate, which both inhibit the initial steps of catalysis, does not ch ange 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 PhAspAT was constructed on the basis of sequence homol ogy with other AspATs. Interestingly, it shows no insertion or extension of loops, but some cavities and a decrease in side chain packing can be obser ved.