CLONING, SEQUENCING AND FUNCTIONAL EXPRESSION OF A CDNA-ENCODING A NADP-DEPENDENT MALIC ENZYME FROM HUMAN LIVER

This work reports the structure of a cDNA (ME) encoding a human malic enzyme (ME) (malate NADP oxidoreductase, EC 1.1.1.40) elucidated by joining several overlapping fragments amplified by PCR from human hepatic cDNA or from cDNA libraries. The full-length cDNA has an open reading frame (ORF) of 1719 bp that encodes a 572-amino-acid protein of 64 113 Da, similar to the native monomeric, cytosolic, NADP-dependent ME isolated from human liver. The comparison of the structure of this cDNA with that of the human mitochondrial NAD(P)-dependent ME (EC 1.1.1.39) shows a homology of 63%, suggesting that these two forms originated from the same gene. The expression of the cDNA in Escherichia coli as a translational fusion (glutathione S-transferase::ME) protein yielded a product of the predicted mass, The recombinant protein shows NADP-dependent malate oxidoreductase activity and is virtually inactive with NAD, It also shows other distinct features of the native cytosolic NADP-dependent ME, like Mn2+ dependence, similar substrate (K-m = 117 mu M) and cofactor affinity (K-m = 2 mu M) constants, and a lack of allosteric regulation. In human proliferative cells, the NADP-dependent ME activity is poorly expressed and barely inducible by thyroid hormones.