The Chlamydomonas reinhardtii chloroplast mutant 68-4PP is phenotypically indistinguishable from wild type at 25°C but fails to grow photosynthetically at 35°C. It had about 30% of the wild-type level of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) holoenzyme and carboxylase activity when grown at 25°C, but less than 15% when grown at 35°C. Pulse-labeling with 35S showed that the decrease in enzyme Ribulose-1,5-bisphosphate (Rbu-P2) carboxylase/oxygenase (EC 4.1.1.39) is a key enzyme in photosynthetic C02 fixation that is assembled from eight chloroplast-encoded large subunits and eight nuclear-encoded small subunits in all plants and most algae (1). It catalyzes both the carboxylation and the oxygenation of Rbu-P2 in the chloroplast and thus initiates the competitive metabolic pathways of photosynthesis and photorespiration, respectively (2). Photorespiration is a nonessential process in plants (3), and the competition of 02 with CO2 at the large-subunit active site (4) ultimately leads to the loss of fixed carbon and a reduction in photosynthetic efficiency (5

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.

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Nebraska 68588-0664 (G.S., R.C.)Pyruvate,orthophosphate (Pi) dikinase (PPDK) is best recognized as a chloroplastic C 4 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 C 3 plants, although it has no known photosynthetic function. Nevertheless, in this report we show that C 3 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 C 4 dikinase. In addition, the kinetics of this process closely resemble the reversible C 4 process, with light-induced dephosphorylation occurring rapidly (Յ15 min) and dark-induced phosphorylation occurring much more slowly (Ն30-60 min). In intact spinach chloroplasts, light-induced dephosphorylation of C 3 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 C 3 -PPDK. Last, we used an in vitro RP assay to directly demonstrate ADP-dependent PPDK phosphorylation in desalted leaf extracts of the C 3 plants Vicia faba and rice (Oryza sativa). We conclude that an RP-like activity mediates the light/dark modulation of PPDK phosphorylation state in C 3 leaves and chloroplasts and likely represents the ancestral isoform of this unusual and key C 4 pathway regulatory "converter" enzyme.Pyruvate,orthophosphate (Pi) dikinase (PPDK; EC 2.7.9.1) is a well-known enzyme of the C 4 photosynthetic pathway where it catalyzes the ATP-and Pidependent formation of phosphoenolpyruvate (PEP), the primary CO 2 acceptor molecule, from pyruvate:Consistent with its being a rate-limiting enzyme in the C 4 cycle, PPDK activity is regulated in a reversible, light-dependent manner so the overall pathway can function optimally in net CO 2 assimilation (Hatch, 1987;Furbank et al., 1997). This posttranslational regulation is conferred by reversible phosphorylation of a "target" Thr residue (Thr-456 in maize [Zea mays] C 4 PPDK) proximal to a catalytically essential (phospho) His (His-458 in maize), with the enzyme being inactive in its threonylphosphorylated state. A single, bifunctional protein kinase/phosphatase, named the PPDK regulatory protein (RP), catalyzes this regulatory phosphorylation/dephosphorylation cycle ( Fig. 1; Burnell and Hatch, 1984, 1985Roeske and Chollet, 1987; Ashton et al., 1990). Along with its target enzyme, RP is specifically localized in the chloroplast stroma of the C 4 mesophyll cell. It is a highly unusual and unique RP in at least three important respects. First, it is bifunctional in that it catalyzes both PPDK in...

). † These authors contributed equally to this work. SummaryPyruvate, orthophosphate dikinase (PPDK) is a ubiquitous, low-abundance metabolic enzyme of undetermined function in C3 plants. Its activity in C3 chloroplasts is light-regulated via reversible phosphorylation of an active-site Thr residue by the PPDK regulatory protein (RP), a most unusual bifunctional protein kinase (PK)/ protein phosphatase (PP). In this paper we document the molecular cloning and functional analysis of the two unique C3 RPs in Arabidopsis thaliana. The first of these, AtRP1, encodes a typical chloroplast-targeted, bifunctional C4-like RP. The second RP gene, AtRP2, encodes a monofunctional polypeptide that possesses in vitro RP-like PK activity but lacks PP activity, and is localized in the cytosol. Notably, the deduced primary structures of these two highly homologous polypeptides are devoid of any canonical subdomain structure that unifies all known eukaryotic and prokaryotic Ser/Thr PKs into one of three superfamilies, despite the direct demonstration that AtRP1 is functionally a member of this group. Instead, these C3 RPs and the related C4 plant homologues encode a conserved, centrally positioned, approximately 260-residue sequence currently described as the 'domain of unknown function 299¢ (DUF 299). We propose that vascular plant RPs form a unique protein kinase family now designated as the DUF 299 gene family.