Abstract
The cDNA fragment of a rice NAD-malic enzyme (OsNAD-ME1) was cloned and constructed into expression vector (pGEX-6p-3). OsNAD-ME1 was successfully expressed as a GST fusion protein in Escherichia coli BL21. The optimal concentration of IPTG for inducement was 1 mmol/L and the optimal culturetemperature was 30°C. The fusion protein was purified by using affinity chromatography with a glutathione sepharose 4B column. After enzymatic cleavage of GST tag, the OsNAD-ME1 recombinant protein was collected for studying its kinetic properties. The optimum pH and temperature for catalytic reaction ofOsNAD-ME1 were pH 6.4 and 35°C, respectively. The kcat value determined at pH 6.4 was 36.38 s-1 and the Km values for NAD+ and malate were 0.10 and 15.98mmol/L, respectively. The maximum activity of OsNAD-ME1 using NADP+ as coenzyme was 64.47% of that using NAD+ as coenzyme. Key words: Enzyme activity, GST fusion protein, kinetic properties, NAD-malic enzyme, Oryza sativa L., purification.
Highlights
Malic enzymes (MEs) widely distributed in nature, have been identified in different organisms, such as bacteria, yeast, fungi, plants, animals and humans
The pT-OsNAD-ME1 plasmid DNA was digested with EcoRI and XhoI, and the digested OsNAD-ME1 cDNA was inserted into pGEX-6p-3 vector (Amersham Pharmacia Biotech) that was digested with the same enzymes
The GST-OsNAD-ME1 fusion protein had a molecular mass of 88 kDa, which was consistent with the sum of the molecular masses of GST (26 kDa) and OsNAD-ME1 (62 kDa) predicted from their nucleotide sequences
Summary
Malic enzymes (MEs) widely distributed in nature, have been identified in different organisms, such as bacteria, yeast, fungi, plants, animals and humans. MEs catalyze the oxidative decarboxylation of L-malate to pyruvate in the presence of cations (typically Mg2+ or Mn2+) with the concomitant reduction of coenzyme NAD+ or NADP+ MEs) belong to the EC 1.1.1.39 subtype, as they are not able to decarboxylate OAA (Maurino et al, 2009). Their amino acid sequences are highly conserved among a large number of organisms, suggesting that MEs have important biological functions. It was considered that plant NADP-malic enzymes (NADP-MEs) were involved in plant defense responses. The mechanism about stress resistance was postulated that the enzyme was implicated in defense-related deposition of lignin and flavonoid by providing NADPH for steps in their biosynthesis pathway requiring reductive power (Casati et al, 1999)
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