A biochemical and molecular characterization of the structure and regulation of chloroplastic NADP-dependent malic enzyme from maize.

摘要

Chloroplast-localized NADP-dependent malic enzyme (EC 1.1.1.40) (NADP-ME) provides a key activity in the C4 photosynthetic pathway. A biochemical and molecular analysis of NADP-ME in maize indicates that the C4 chloroplastic and C3 cytosolic isozymes are encoded by separate nuclear loci. Chloroplastic NADP-ME has a higher affinity for malate and becomes active at a higher pH than cytosolic NADP-ME. The catalytic properties of C4 NADP-ME are such that it can function in illuminated chloroplasts yet be inactive in the dark, minimizing potential interference with the metabolic role of the cytosolic isozyme.; Amino acid sequence comparison to bacterial NAD-ME and cytosolic NADP-ME from rat, mouse and bean indicate extensive sequence similarity, supporting the hypothesis that chloroplastic NADP-ME was derived from a constitutive eukaryotic form of the enzyme. Two conserved regions have been identified as putative dinucleotide binding sites. These sites may act independently or form a single site via protein folding and the formation of a conserved disulfide bond.; The presence of an intron near the processing site of the precursor protein suggests that chloroplastic NADP-ME was formed by the addition of a transit peptide to the 5{dollar}spprime{dollar} end of an existing NADP-ME locus. The transit peptide has the general characteristics typical of known chloroplast stroma transit peptides, although there is no conserved homology. Codon usage in the 5{dollar}spprime{dollar} end of the chloroplastic NADP-ME gene is strongly biased toward C or G in the third position. This pattern of codon biasing is found in the genes encoding the other C4 pathway enzymes and several highly expressed photosynthetic proteins in maize.; Accumulation of NADP-ME protein during maize leaf development corresponds to message accumulation indicating that regulation is either transcriptional or via transcript stability. C4 NADP-ME is leaf-specific and accumulates in response to positional cues in the bundle sheath cells surrounding developing veins. The developmental timing of message appearance is independent of illumination. Light enhances overall levels of message. The other C4 pathway enzymes respond similarly with their own characteristic cellular localization, suggesting that the pathway may have common regulatory mechanisms yet to be elucidated.