Chris J. Chastain

Functional evolution of C4 pyruvate, orthophosphate dikinase

Pyruvate,orthophosphate dikinase (PPDK) plays a controlling role in the PEP-regeneration phase of the C(4) photosynthetic pathway. Earlier studies have fully documented its biochemical properties and its post-translational regulation by the PPDK regulatory protein (PDRP). However, the question of its evolution into the C(4) pathway has, until recently, received little attention. One assumption concerning this evolution is that changes in catalytic and regulatory properties of PPDK were necessary for the enzyme to fulfil its role in the C(4) pathway. In this study, the functional evolution of PPDK from its ancient origins in the Archaea to its ascension as a photosynthetic enzyme in modern C(4) angiosperms is reviewed. This analysis is accompanied by a comparative investigation into key catalytic and regulatory properties of a C(3) PPDK isoform from Arabidopsis and the C(4) PPDK isoform from Zea mays. From these analyses, it is proposed that PPDK first became functionally seated in C(3) plants as an ancillary glycolytic enzyme and that its transition into a C(4) pathway enzyme involved only minor changes in enzyme properties per se.

Pyruvate,Orthophosphate Dikinase in Leaves and Chloroplasts of C3 Plants Undergoes Light-/Dark-Induced Reversible Phosphorylation

Pyruvate,orthophosphate (Pi) dikinase (PPDK) is best recognized as a chloroplastic C4 cycle enzyme. As one of the key regulatory foci for controlling flux through this photosynthetic pathway, it is strictly and reversibly regulated by light. This light/dark modulation is mediated by reversible phosphorylation of a conserved threonine residue in the active-site domain by the PPDK regulatory protein (RP), a bifunctional protein kinase/phosphatase. PPDK is also present in C3 plants, although it has no known photosynthetic function. Nevertheless, in this report we show that C3 PPDK in leaves of several angiosperms and in isolated intact spinach (Spinacia oleracea) chloroplasts undergoes light-/dark-induced changes in phosphorylation state in a manner similar to C4 dikinase. In addition, the kinetics of this process closely resemble the reversible C4 process, with light-induced dephosphorylation occurring rapidly (≤15 min) and dark-induced phosphorylation occurring much more slowly (≥30–60 min). In intact spinach chloroplasts, light-induced dephosphorylation of C3 PPDK was shown to be dependent on exogenous Pi and photosystem II activity but independent of electron transfer from photosystem I. These in organello results implicate a role for stromal pools of Pi and adenylates in regulating the reversible phosphorylation of C3-PPDK. Last, we used an in vitro RP assay to directly demonstrate ADP-dependent PPDK phosphorylation in desalted leaf extracts of the C3 plantsVicia faba and rice (Oryza sativa). We conclude that an RP-like activity mediates the light/dark modulation of PPDK phosphorylation state in C3 leaves and chloroplasts and likely represents the ancestral isoform of this unusual and key C4 pathway regulatory “converter” enzyme.

Regulation of pyruvate, orthophosphate dikinase by ADP-/Pi-dependent reversible phosphorylation in C3 and C4 plants☆

Abstract Pyruvate, orthophosphate dikinase (PPDK, E.C. 2.7.9.1) is a cardinal carbon-assimilating, stromal enzyme of the C 4 photosynthetic pathway. Like several other photosynthetic pathway enzymes, its activity is strictly and reversibly regulated by light. This regulation is conferred by the PPDK regulatory protein (RP), a bifunctional protein kinase/phosphatase that catalyzes the ADP-/Pi-dependent, reversible phosphorylation of an active-site threonine residue. In this minireview, we highlight how plastidic PPDK in leaves and developing seeds of C 3 plants is regulated in an identical manner as C 4 PPDK via a putative C 3 -RP isoform. Additionally, we also detail the progress in research concerning C 4 RP, since this highly unusual regulatory enzyme was last reviewed nearly two decades ago.

Site‐directed mutagenesis of maize recombinant C4‐pyruvate,orthophosphate dikinase at the phosphorylatable target threonine residue

A key regulatory enzyme of the C4‐photosynthetic pathway is stromal pyruvate,orthophosphate dikinase (PPDK, EC 2.7.9.1). As a pivotal enzyme in the C4 pathway, it undergoes diurnal light–dark regulation of activity which is mediated by a single bifunctional regulatory protein (RP). RP specifically inactivates PPDK in the dark by an ADP‐dependent phosphorylation of an active‐site Thr residue (Thr‐456 in maize). Conversely, RP activates inactive PPDK in the light by phosphorolytic dephosphorylation of this target Thr‐P residue. We have employed a His‐tagged maize recombinant C4 PPDK for directed mutagenesis of this active‐site regulatory Thr. Three such mutants (T456V, T456S, T456D) were analyzed with respect to overall catalysis and regulation by exogenous maize RP. Substitution with Val and Ser at this position does not affect overall catalysis, whereas Asp abolishes enzyme activity. With respect to regulation by RP, it was found that Ser can effectively substitute for the wild‐type Thr residue in that mutant enzyme is phosphorylated and inactivated by RP. The T456V mutant, however, could not be phosphorylated and was, thus, resistant to ADP‐dependent inactivation by RP.

The pyruvate, orthophosphate dikinase regulatory proteins of Arabidopsis are both bifunctional and interact with the catalytic and nucleotide-binding domains of pyruvate, orthophosphate dikinase

Pyruvate orthophosphate dikinase (PPDK) is a key enzyme in C(4) photosynthesis and is also found in C(3) plants. It is post-translationally modified by the PPDK regulatory protein (RP) that possesses both kinase and phosphotransferase activities. Phosphorylation and dephosphorylation of PPDK lead to inactivation and activation respectively. Arabidopsis thaliana contains two genes that encode chloroplastic (RP1) and cytosolic (RP2) isoforms of RP, and although RP1 has both kinase and phosphotransferase activities, to date RP2 has only been shown to act as a kinase. Here we demonstrate that RP2 is able to catalyse the dephosphorylation of PPDK, although at a slower rate than RP1 under the conditions of our assay. From yeast two-hybrid analysis we propose that RP1 binds to the central catalytic domain of PPDK, and that additional regions towards the carboxy and amino termini are required for a stable interaction between RP2 and PPDK. For 21 highly conserved amino acids in RP1, mutation of 15 of these reduced kinase and phosphotransferase activity, while mutation of six residues had no impact on either activity. We found no mutant in which only one activity was abolished. However, in some chimaeric fusions that comprised the amino and carboxy termini of RP1 and RP2 respectively, the kinase reaction was severely compromised but phosphotransferase activity remained unaffected. These findings are consistent with the findings that both RP1 and RP2 modulate reversibly the activity of PPDK, and possess one bifunctional active site or two separate sites in close proximity.

Maize recombinant C4-pyruvate,orthophosphate dikinase: Expression in Escherichia coli, partial purification, and characterization of the phosphorylatable protein.

The gene for C4-pyruvate,orthophosphate dikinase (PPDK) from maize (Zea mays) was cloned into an Escherichia coli expression vector and recombinant PPDK produced in E. coli cells. Recombinant enzyme was found to be expressed in high amounts (5.3 U purified enzyme-activity liter-1 of induced cells) as a predominantly soluble and active protein. Biochemical analysis of partially purified recombinant PPDK showed this enzyme to be equivalent to enzyme extracted from illuminated maize leaves with respect to (i) molecular mass, (ii) specific activity, (iii) substrate requirements, and (iv) phosphorylation/inactivation by its bifunctional regulatory protein.

Chapter 15 Structure, Function, and Post-translational Regulation of C4 Pyruvate Orthophosphate Dikinase

Pyruvate orthophosphate dikinase is a cardinal enzyme of the C4 pathway. Its role in C4 photosynthesis is to catalyze the regeneration of PEP, the primary carboxylation substrate from pyruvate, Pi, and ATP in the chloroplast stroma of leaf-mesophyll cells. It is the most abundant of C4 enzymes, comprising up to 10% of the soluble protein of C4 leaves, and thus may exert a limitation on the rate of CO2 assimilation into the C4-cycle. Studies dating back to the 1970s documented its biochemical properties as related to its role in C4 photosynthetic process. Later studies originating in the early 1980s discovered how the enzyme is regulated in a light/dark manner by reversible phosphorylation of an active-site threonine. A bifunctional protein kinase/protein phosphatase with unprecedented properties, the PPDK Regulatory Protein (RP), was identified as the enzyme catalyzing this reversible phosphorylation event. However, the gene encoding this unusual enzyme had eluded cloning for some two decades until modern cloning methods allowed its recent isolation from maize. Although the enzyme properties of C4-PPDK are well understood, the molecular basis of its post-translational light/dark regulation by RP is poorly understood. Because of the significance of PPDK regulation to the C4-photosynthetic process, this chapter addresses the current state-of-knowledge on how C4-PPDK is post-translationally regulated by its companion regulatory enzyme, RP. This includes proposed models that describe how phosphorylation of PPDK by RP leads to complete inactivation of enzyme activity and the mechanism regulating the direction of RP’s opposing PPDK-dephosphorylation and PPDK-phosphorylation activities. Also reviewed are the recent bioinformatic analyses of the RP polypeptide primary structure. These revealed that vascular plant RP represents a fundamentally new and novel kind of protein kinase with evolutionary origins in PPDK-containing anaerobic bacteria.

Implementing the recommended curriculum in biochemistry and molecular biology at a regional comprehensive university through a biology/chemistry double major: The minnesota state university moorhead experience*.

Minnesota State University Moorhead (MSUM) is a regional comprehensive university that is part of the Minnesota State Colleges and Universities (MnSCU) system. The current student population consists of ∼7,600 full‐ and part‐time students who are enrolled in one of 135 majors that lead to baccalaureate degrees. MSUM is committed to excellence in science teaching and research for undergraduates. It is an institutional member of the Council on Undergraduate Research and has three faculty members participating in Project Kaleidoscope (PKAL) Faculty for the 21st Century. Fourteen years ago, MSUM renewed its effort to have faculty participate in active research. All science faculty members hired since that time have been required to establish research programs. The primary purpose for the faculty engaging in ongoing research projects is to involve undergraduates in a meaningful research experience, thus training these students to become scientists.

Maize leaf PPDK regulatory protein isoform-2 is specific to bundle sheath chloroplasts and paradoxically lacks a Pi-dependent PPDK activation activity

In the maize C4 pathway, PDRP1 is known to regulate C4 PPDK activity in mesophyll chloroplasts. A second PDRP isoform, PDRP2, is shown to be the apparent regulator of C4 PPDK in bundle sheath chloroplasts.