Identification of Domains Involved in Tetramerization and Malate Inhibition of Maize C4-NADP-Malic Enzyme

Abstract

C 4 photosynthetic NADP-malic enzyme (ME) has evolved from non-C 4 isoforms and gained unique kinetic and structural properties during this process. To identify the domains responsible for the structural and kinetic differences between maize C 4 and non-C 4 -NADP-ME several chimeras between these isoforms were constructed and analyzed. By using this approach, we found that the region flanked by amino acid residues 102 and 247 is critical for the tetrameric state of C 4 -NADP-ME. In this way, the oligomerization strategy of these NADP-ME isoforms differs markedly from the one that present non-plant NADP-ME with known crystal structures. On the other hand, the region from residue 248 to the C-terminal end of the C 4 isoform is involved in the inhibition by high malate concentrations at pH 7.0. The inhibition pattern of the C 4 -NADP-ME and some of the chimeras suggested an allosteric site responsible for such behavior. This pH-dependent inhibition could be important for regulation of the C 4 isoform in vivo, with the enzyme presenting maximum activity while photosynthesis is in progress.

Plastidial NADP-malic enzymes from grasses: Unraveling the way to the C4 specific isoforms

“…3a), which was also previously described for ZmC 4 -NADP-ME [9]. On the other hand, in the case of ZmnonC 4 -and SbnonC 4 -NADP-ME, weak malate inhibition was observed at pH 7.0 (Fig.…”

“…At this pH, the sorghum photosynthetic NADP-ME (SbC 4 -NADP-ME) displayed significant higher catalytic efficiencies for both NADP and malate than the non-photosynthetic counterpart (SbnonC 4 -NADP-ME, Table 1). This is also verified when comparing maize photosynthetic and nonphotosynthetic NADP-MEs (Table 1), as previously indicated [9].…”

“…Unfortunately, this approach was unsuccessful with ZmnonC 4 -NADP-ME due to the low yield of the purification from maize roots [26]. In previous works with this enzyme, the mature recombinant enzyme was obtained considering the cleavage site between residues 40 and 41 predicted in silico [34,9]. Nevertheless, recent results indicate that the transit peptide is processed in vivo near the residue analogous to number 62 of ZmC 4 -NADP-ME (unpublished results).…”

“…Malic enzyme is represented by a multi-member family of plastidial, cytosolic and mitochondrial isoforms in maize [11,9,12,34,24]. The enzyme that participates in the C 4 cycle (ZmC 4 -NADP-ME, L-malate:NADP oxidoreductase, E.C.…”

“…1.1.1.40, nadpme-IVa gene named by Christin et al [7]) are both plastidial but differ in expression pattern, kinetics and structure. The photosynthetic enzyme is inhibited by high malate concentrations at pH 7.0 and assembles as tetramers, but the non-photosynthetic isoform is not inhibited by malate and assembles as dimers [11,9,34]. Moreover, ZmC 4 -NADP-ME activity, but not ZmnonC 4 -NADP-ME activity, is redox-modulated [2].…”