Three different and tissue-specific NAD-malic enzymes generated by alternative subunit association in Arabidopsis thaliana.
The Arabidopsis thaliana genome contains two genes encoding the mitochondrial NAD-malic enzyme (NAD-ME), NAD-ME1 (At2g13560) and NAD-ME2 (At4g00570). The characterization of recombinant NAD-ME1 and -2 indicated that both enzymes assemble as active homodimers; however, a heterodimeric enzyme (NAD-MEH) can also be detected by electrophoretic studies. To analyze the metabolic contribution of each enzymatic entity, NAD-MEH was obtained by a co-expression-based recombinant approach, and its kinetic and regulatory properties were analyzed. The three NAD-MEs show similar kinetic properties, although they differ in the regulation by several metabolic effectors. In this regard, whereas fumarate activates NAD-ME1 and CoA activates NAD-ME2, both compounds act synergistically on NAD-MEH activity. The characterization of two chimeric enzymes between NAD-ME1 and -2 allowed specific domains of the primary structure, which are involved in the differential allosteric regulation, to be identified. NAD-ME1 and -2 subunits showed a distinct pattern of accumulation in the separate components of the floral organ. In sepals, the NAD-ME1 subunit is present at a slightly higher proportion than the NAD-ME2 subunit, and thus, NAD-MEH and NAD-ME1 act in concert in this tissue. On the other hand, NAD-ME2 is the only isoform present in anthers. In view of the different properties of NAD-ME1, -2, and -H, we suggest that mitochondrial NAD-ME activity may be regulated by varying native association in vivo, rendering enzymatic entities with distinct allosteric regulation to fulfill specific roles. The presence of three different NAD-ME entities, which originate by alternative associations of two subunits, is suggested to be a novel phenomenon unique to plant mitochondria.
Tronconi
Marcos A
MA
Centro de Estudios Fotosintéticos y Bioquímicos, Universidad Nacional de Rosario, Rosario, Argentina.
Maurino
Verónica G
VG
Andreo
Carlos S
CS
Drincovich
María F
MF
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003065:Coenzyme A / Q000494:pharmacology; D017931:DNA Primers / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D005650:Fumarates / Q000494:pharmacology; D017343:Genes, Plant; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology; D008954:Models, Biological; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D029681:Arabidopsis Proteins; D017931:DNA Primers; D005650:Fumarates; D007527:Isoenzymes; D021122:Protein Subunits; D011994:Recombinant Proteins; C032005:fumaric acid; D008291:Malate Dehydrogenase; C046446:malate dehydrogenase-(oxaloacetate-decarboxylating) (NAD+); D003065:Coenzyme A