Heterologous gene expression and characterization of recombinant aspartate aminotransferase from Geobacillus thermopakistaniensis.

Aspartate aminotransferase catalyzes the transfer of an amino group from l-aspartate to α-oxoglutarate. A gene encoding aspartate aminotransferase, ASTGt, from Geobacillus thermopakistaniensis was cloned and expressed in Escherichia coli. The purified recombinant ASTGt exhibited highest activity at 65 °C and pH 7.0. The activity was dependent on pyridoxal phosphate but not on any metal ions. Stoichiometry of purified ASTGt demonstrated that 0.1 pyridoxal phosphate was attached per subunit of the enzyme. Determination of molecular weight by gel filtration chromatography indicated that ASTGt existed in a dimeric form in solution. Thermostability experiments showed no significant change in activity even after 16 h incubation at 65 °C. ASTGt exhibited apparent Vmax and Km values of 120 µmol min-1 mg-1 and 1.5 mM, respectively, against l-aspartate. Substrate specificity experiments indicated the highest relative activity against aspartate (100%) followed by tyrosine (27%) and proline (16%). To the best of our knowledge, this is the first report on cloning and characterization of an AST from genus Geobacillus.

Heterologous gene expression and characterization of TK2246, a highly active and thermostable plant type l-asparaginase from Thermococcus kodakarensis.

The genome sequence of the hyperthermophilic archaeon Thermococcus kodakarensis contains two putative genes, TK1656 and TK2246, annotated as l-asparaginases. TK1656 has been reported previously. The current report is focused on TK2246, a plant-type l-asparaginase, which consists of 918 nucleotides corresponding to a polypeptide of 306 amino acids. The gene was cloned, expressed in Escherichia coli and the purified gene product was used to determine the properties of the recombinant enzyme. TK2246 was optimally active at 85 °C and pH 7.0 with a specific activity of 767 µmol min-1 mg-1 towards l-asparagine. The enzyme exhibited a 10% activity towards d-asparagine as compared to 100% against l-asparagine. No detectable activity was observed towards l- or d-glutamine. Half-life of the enzyme was nearly 18 h at 85 °C. TK2246 exhibited apparent Km and Vmax values of 3.1 mM and 833 µmol min-1 mg-1, respectively. Activation energy of the reaction, determined from the Arrhenius plot, was 28.3 kJ mol-1. To the best of our knowledge, this is the first characterization of a plant-type l-asparaginase from class Thermococci of phylum Euryarchaeota.

Pcal_0970: an extremely thermostable L-asparaginase from Pyrobaculum calidifontis with no detectable glutaminase activity.

The genome sequence of Pyrobaculum calidifontis contains two open reading frames, Pcal_0144 and Pcal_0970, exhibiting homology with L-asparaginases. In search of a thermostable L-asparaginase with no glutaminase activity, we have cloned and expressed the gene encoding Pcal_0970 in Escherichia coli. Recombinant Pcal_0970 was produced in insoluble and inactive form which was solubilized and refolded into enzymatically active form. The refolded Pcal_0970 showed the highest activity at or above 100 °C. Optimum pH for the enzyme activity was 6.5. Addition of divalent metal cations or EDTA had no significant effect on the activity. The enzyme was capable of hydrolyzing D-asparagine with a 20% activity as compared to 100% with L-asparagine. Pcal_0970 did not show any detectable activity when L-glutamine or D-glutamine was used as substrate. Pcal_0970 exhibited a Km value of 4.5 ± 0.4 mmol/L and Vmax of 355 ± 13 µmol min-1 mg-1 towards L-asparagine. The activation energy, from the linear Arrhenius plot, was determined as 39.9 ± 0.6 kJ mol-1. To the best of our knowledge, Pcal_0970 is the most thermostable L-asparaginase with a half-life of more than 150 min at 100 °C and this is the first report on characterization of an L-asparaginase from phylum Crenarchaeota.

Structure and function of the thermostable L-asparaginase from Thermococcus kodakarensis.

L-Asparaginases catalyse the hydrolysis of asparagine to aspartic acid and ammonia. In addition, L-asparaginase is involved in the biosynthesis of amino acids such as lysine, methionine and threonine. These enzymes have been used as chemotherapeutic agents for the treatment of acute lymphoblastic leukaemia and other haematopoietic malignancies since the tumour cells cannot synthesize sufficient L-asparagine and are thus killed by deprivation of this amino acid. L-Asparaginases are also used in the food industry and have potential in the development of biosensors, for example for asparagine levels in leukaemia. The thermostable type I L-asparaginase from Thermococcus kodakarensis (TkA) is composed of 328 amino acids and forms homodimers in solution, with the highest catalytic activity being observed at pH 9.5 and 85°C. It has a Km value of 5.5 mM for L-asparagine, with no glutaminase activity being observed. The crystal structure of TkA has been determined at 2.18 Šresolution, confirming the presence of two α/ß domains connected by a short linker region. The N-terminal domain contains a highly flexible ß-hairpin which adopts `open' and `closed' conformations in different subunits of the solved TkA structure. In previously solved L-asparaginase structures this ß-hairpin was only visible when in the `closed' conformation, whilst it is characterized with good electron density in all of the subunits of the TkA structure. A phosphate anion resides at the active site, which is formed by residues from both of the neighbouring monomers in the dimer. The high thermostability of TkA is attributed to the high arginine and salt-bridge content when compared with related mesophilic enzymes.

TK1656, a thermostable l-asparaginase from Thermococcus kodakaraensis, exhibiting highest ever reported enzyme activity.

Two L-asparaginase homologs, TK1656 and TK2246, have been found in the genome of Thermococcus kodakaraensis. The gene encoding TK1656 consists of 984 nucleotides corresponding to a polypeptide of 328 amino acids. To examine the properties of TK1656, the structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. TK1656 exhibited high asparaginase activity (2350 U mg⁻¹) but no glutaminase activity. The enzyme also displayed the D-asparaginase activity but 50% to that of L-asparaginase. The highest activity was observed at 85°C and pH 9.5. TK1656 catalyzed the conversion of L-asparagine to L-aspartatic acid and ammonia following Michaelise-Menten kinetics with a K(m) and V(max) values of 5.5 mM and 3300 mmol min⁻¹ mg⁻¹, respectively. The activation energy from the linear Arrhenius plot was found to be 58 kJ mol⁻¹. Unfolding studies suggested that urea could not induce complete unfolding and inactivation of TK1656 even at a concentration 8 M; however, in the presence of 4 M guanidine hydrochloride enzyme structure was unfolded with complete loss of enzyme activity.