Cloning of a 1‐pyrroline‐5‐carboxylate dehydrogenase from Taiwanofungus camphorata (583.1)

Abstract

A cDNA (1890 bp) encoding a putative 1‐pyrroline‐5‐carboxylate dehydrogenase (P5CD) was cloned from Taiwanofungus camphorata. The DNA sequence encodes a protein of 547 amino acid residues with calculated molecular mass of 59.4 kDa. The deduced amino acid sequence is conserved among the reported P5CDs. To characterize the T. camphorata P5CD, the coding region was subcloned into an expression vector pET‐20b(+) and transformed into E. coli Rosetta (DE3). The recombinant His6‐tagged TcP5CD was expressed and purified by Ni2+‐nitrilotriacetic acid Sepharose. The purified enzyme showed a major band of ~59.4 kDa by 12 % SDS‐PAGE. The Michaelis constant (KM) value for 3,5‐dimethoxybenzaldehyde with NAD+ as cofactor was 0.56 mM. The enzyme’s half‐life of deactivation at 45oC was 4.4 min, and its thermal inactivation rate constant kd was 1.23 x 10‐2 min‐1. The enzyme was most active at pH 7.0. The enzyme’s preferred substrate is veratraldehyde. It can also use other aldehyde as substrates including acetaldehyde and propionaldehyde. Grant Funding Source: National Science Council of the Republic of China under grant NSC95‐2313‐B‐019‐007 to C‐T. L.