Full-length cDNAs encoding the α- and β-subunits and a truncated mutant subunit of the Chlorella sorokiniana NADP-GDH isozymes were constructed and expressed in Escherichia coli cells. The kinetic and thermal stability properties of the resultant homohexamers were examined. The electrophoretic mobility of the recombinant α- and β-subunits was identical to that of the native subunits as determined by immunoblotting. The homohexamers were purified by anion-exchange and gel-filtration chromatography. The α- and β-homohexamers that were synthesized in the bacterial cells were shown to have similar Michaelis constants for their substrates as previously shown after synthesis in C. sorokiniana cells (Bascomb and Schmidt, 1987). The α homohexamer synthesized in the bacterium was allosteric with respect to NADPH but to a lesser degree than when isolated from the alga. The mutant homohexamer was composed of subunits that were truncated by 40 amino acids at their N-termini. This mutant isozyme was kinetically similar to the larger, anabolic α-homohexamer, but it did not display the allosteric response to NADPH shown by the α-homohexamer. The three isozymes had significant thermal tolerance and were stable at 50 °C. The temperature optimum for catalytic activity for the α- and β-homohexamers was 60 °C, and 65 °C for the Δ40N homohexamer. This study demonstrated that most of the kinetic properties of the Chlorella sorokiniana NADP-GDH isozymes were retained after their synthesis in a heterologous system, and that the distinctive N-terminal domains of these isozymes have dramatic effects on their biochemical characteristics.
Plant Molecular Biology – Springer Journals
Published: Oct 7, 2004
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