Posttranslational Modification of Maize Chloroplast Pyruvate Orthophosphate Dikinase Reveals the Precise Regulatory Mechanism of Its Enzymatic Activity

Abstract

In C 4 plants, pyruvate orthophosphate dikinase (PPDK) activity is tightly dark/light regulated by reversible phosphorylation of an active-site threonine (Thr) residue; this process is catalyzed by PPDK regulatory protein (PDRP). Phosphorylation and dephosphorylation of PPDK lead to its inactivation and activation, respectively. Here, we show that light intensity rather than the light/dark transition regulates PPDK activity by modulating the reversible phosphorylation at Thr-527 (previously termed Thr-456) of PPDK in maize (Zea mays). The amount of PPDK (unphosphorylated) involved in C 4 photosynthesis is indeed strictly controlled by light intensity, despite the high levels of PPDK protein that accumulate in mesophyll chloroplasts. In addition, we identified a transit peptide cleavage site, uncovered partial amino-terminal acetylation, and detected phosphorylation at four serine (Ser)/Thr residues, two of which were previously unknown in maize. In vitro experiments indicated that Thr-527 and Ser-528, but not Thr-309 and Ser-506, are targets of PDRP. Modeling suggests that the two hydrogen bonds between the highly conserved residues Ser-528 and glycine-525 are required for PDRP-mediated phosphorylation of the active-site Thr-527 of PPDK. Taken together, our results suggest that the regulation of maize plastid PPDK isoform (C 4 PPDK) activity is much more complex than previously reported. These diverse regulatory pathways may work alone or in combination to fine-tune C 4 PPDK activity in response to changes in lighting.

Transgenic Perturbation of the Decarboxylation Phase of Crassulacean Acid Metabolism Alters Physiology and Metabolism But Has Only a Small Effect on Growth

“…3B) is consistent with PPDK phosphorylation playing a pivotal role in the LD temporal coordination of CAM, and correlates with the LD dephosphorylation/phosphorylation of PPDK during C 4 photosynthesis (Burnell and Chastain, 2006;Chen et al, 2014). However, to our knowledge, this is the first report of LD phosphoregulation of PPDK in a CAM species.…”

Structural Basis of Reversible Phosphorylation by Maize Pyruvate Orthophosphate Dikinase Regulatory Protein

“…Phosphorylation and dephosphorylation of residue Thr-527 of PPDK lead to its inactivation and activation, respectively. This process is accomplished by a single, bifunctional protein, namely PPDK regulatory protein (PDRP; Burnell and Hatch, 1985), and the process is light intensity dependent (Hatch and Slack, 1969;Chen et al, 2014b). PDRP is an unusual enzyme in three respects.…”

Reduction of pyruvate orthophosphate dikinase activity is associated with high temperature-induced chalkiness in rice grains

“…After Fukayama et al (2001) firstly reported the existence of an internal PPDK regulatory protein (PDRP) in rice, Chastain et al (2002Chastain et al ( , 2006 confirmed that PPDK underwent a rapid, posttranslational down regulation in activity via PDRP catalyzed Thr527 phosphorylation of PPDK in developing rice grains. Recently, Chen et al (2014) found that Ser528 was another target of PDRP. But it is not as important as Thr527 in phosphorylation because of the space steric effect.…”

“…It is a bifunctional protein that catalyzes both PPDK inactivation (phosphorylation) and activation (dephosphorylation) (Chastain and Chollet, 2003). The most recent research shows that changes in light intensity, rather than light/dark transition, regulates the activity of PPDK in maize (Zea mays) leaves via reversible phosphorylation at Thr527 (Chen et al, 2014).…”