Duck liver ‘malic’ enzyme. Expression in Escherichia coli and characterization of the wild-type enzyme and site-directed mutants

Abstract

A cDNA for duck liver 'malic' enzyme (EC 1.1.1.40) was subcloned into pUC-8, and the active enzyme was expressed in Escherichia coli TG-2 cells as a fusion protein including a 15-residue N-terminal leader from beta-galactosidase coded by the lacZ' gene. C99S and R70Q mutants of the enzyme were generated by the M13 mismatch technique. The recombinant enzymes were purified to near homogeneity by a simple two-step procedure and characterized relative to the enzyme isolated from duck liver. The natural duck enzyme has a subunit molecular mass of approx. 65 kDa, and the following kinetic parameters for oxidative decarboxylation of L-malate at pH 7.0: Km NADP+ (4.6 microM); Km L-malate (73 microM); kcat (160 s-1); Ka (2.4 microM) and Ka' (270 microM), dissociation constants of Mn2+ at 'tight' (activating) and 'weak' metal sites; and substrate inhibition (51% of kcat. at 8 mM-L-malate). Properties of the E. coli-derived recombinant wild-type enzyme are indistinguishable from those of the natural duck enzyme. Kinetic parameters of the R70Q mutant are relatively unaltered, indicating that Arg-70 is not required for the reaction. The C99S mutant has unchanged Km for NADP+ and parameters for the 'weak' sites (i.e. inhibition by L-malate, Ka'); however, kcat. decreased 3-fold and Km for L-malate and Ka each increased 4-fold, resulting in a catalytic efficiency [kcat./(Km NADP+ x Km L-malate x Ka)] equal to 3.7% of the natural duck enzyme. These results suggest that the positioning of Cys-99 in the sequence is important for proper binding of L-malate and bivalent metal ions.

Characterization of cytosolic malic enzyme in human tumor cells

“…Most of the amino acid changes are conservative exchanges. The cytosolic duck ME [12] is more distantly related to the human isoform; the identity is 77%. Most of the differences between the duck and the human and rat ME are clustered in the N-and C-terminal regions of the protein, with the central portion showing the highest degree of conservation.…”

“…Our approach to cloning was a plaque hybridization under reduced stringency using a duck liver ME cDNA [12] as a probe. Of a total of 10 6 phage plaques, 20 were positive after plaque purification.…”

“…2.3. Cloning, sequencing and expression of human cytosolic ME A human fat cell cDNA library (Clontech 1108b) was screened with a cDNA clone of duck ME [12] using radioactive plaque hybridization at reduced stringency [13]. Inserts of positive phages were re-cloned into Bluescript KS(+) using standard procedures [13].…”

Crystallization and preliminary X-ray diffraction analysis of malic enzyme from pigeon liver

“…1.1.1.40) catalyzes the divalent metal ion dependent oxidative decarboxylation of l-malate to yield pyruvate and CO 2 with concomitant reduction of NADP + to NADPH, l-malate NADP Mg 2 or Mn 2 pyruvate CO 2 NADPH H X The malic enzyme was ®rst discovered in pigeon liver in 1947 (Ochoa et al, 1947). Since then, numerous malic enzymes have been found in various organisms such as bacteria (Kobayashi et al, 1989), plants (Rothermel & Nelson, 1989;Westhoff & Borsch, 1990) and higher animals (Bagchi et al, 1987;Hsu et al, 1992;Loeber et al, 1991). In cytosol, the major physiological function of the malic enzyme is to provide NADPH for the de novo biosynthesis of long-chain fatty acids (Goodridge et al, 1989).…”

Transcriptional Regulation of the Schizosaccharomyces pombe Malic Enzyme Gene, mae2

“…Molecular analysis of the S. pombe malic mae2 gene showed a high degree of homology with malic enzymes from various organisms (6). Eight highly conserved regions were identified in malic enzymes, including the binding sites for L-malate and the dinucleotide co-factors NAD(P) ϩ (7,8). Although the secondary structure of malic enzymes is highly conserved, the coenzyme specificity (NAD ϩ or NADP ϩ ) and cellular localization (cytosolic or mitochondrial) are strongly linked to their regulation and metabolic function (9).…”