Chapter 15 Structure, Function, and Post-translational Regulation of C4 Pyruvate Orthophosphate Dikinase Chris J. Chastain Department of Biosciences, Minnesota State University-Moorhead , Moorhead , MN 56563 , USA Summary .............................................................................................................................................................. 301 I. Introduction .................................................................................................................................................... 302 A. Role of PPDK in C 4 Plants ...................................................................................................................... 302 B. PPDK Enzyme Properties ...................................................................................................................... 302 1. Catalysis as Related to Structure ..................................................................................................... 302 2. Oligomeric Structure and Tetramer Dissociation at Cool Temperatures ........................................... 304 3. Substrate Km s for C 4 PPDK .............................................................................................................. 304 C. PPDK as a Rate-Limiting Enzyme of the C 4 Pathway ............................................................................ 304 II. Post-translational Regulation of C 4 PPDK ..................................................................................................... 305 A. Light/Dark Regulation of C 4 PPDK Activity by Reversible Phosphorylation ............................................ 305 1. Discovery of the PPDK Regulatory Protein, RP ............................................................................... 305 2. PPDK RP: Enzyme Properties ......................................................................................................... 305 3. The PPDK Phosphoryl-Inactivation Mechanism .............................................................................. 306 4. Regulation of RP’s Opposing Activities ............................................................................................ 307 B. Other Post-translational Components Governing PPDK Activity In Vivo ................................................ 310 III. Functional and Bioinformatic Analysis of Cloned Maize C 4 and Arabidopsis C 4-Like PPDK-Regulatory Protein ............................................................................................................................. 310 A. Cloning of RP from Maize and Arabidopsis ............................................................................................ 310 B. Functional Properties of Recombinant Maize C 4- and Arabidopsis C 4-Like RP ...................................... 311 C. Bioinformatic Analysis of RP Primary Amino Acid Sequence ................................................................ 312 1. RP Is Highly Conserved in C 3 and C 4 Plants .................................................................................... 312 2. RP Represents a Fundamentally New Structural Class of Regulatory Protein Kinase .................... 312 IV. Future Directions ........................................................................................................................................... 313 Acknowledgments ................................................................................................................................................ 313 References ........................................................................................................................................................... 313 Summary 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 assimila- tion 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. Author for Correspondence, e-mail: [email protected] Agepati S. Raghavendra and Rowan F. Sage (eds.), C 4 Photosynthesis and Related CO 2 Concentrating Mechanisms, pp. 301–315. 301 © Springer Science+Business Media B.V. 2011 302 Chris J. Chastain 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 regu- latory 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. I. Introduction (PEP) in the stroma of leaf-mesophyll cell chlo- roplasts: Pyruvate orthophosphate dikinase (PPDK, E.C. 2.7.9.1) is an ancient enzyme found in a diverse group of microorganisms that includes the archea (Tjaden et al., 2006 ) , eubacteria (Pocalyko et al., 1990 ; Herzberg et al., 1996 ) , amitochondri- ate protozoa (Bringaud et al., 1998 ) and green algae (Chastain and Chollet, 2003 ) . It is absent in cyanobacteria and metazoans, but is evidently present in lower fungi (Marshall et al., 2001) . Its Although its catalysis is freely reversible, the evolution in (C ) plants and recruitment into the reaction is maintained in the PEP forming direc- 3 tion by the abundant pyrophosphatase and ade- C4 pathway has been proposed to be the result of modifications of the gene promoter to confer cell nylate kinase activities in this organelle as well specific expression (Sheen, 1991 ) . In this regard, as the physiochemical factors prevailing during illumination such as stromal alkaline pH (Jenkins its transcriptional regulation, as with other key C4 enzymes, is an important overall component of C and Hatch, 1985 ; Ashton et al., 1990 ) . It is the sole 4 PEP regenerating mechanism for photosynthetic photosynthesis regulation. This aspect of PPDK + regulation is covered in Chapter 12 . This chapter PEP carboxylase (PEPc) fixation in NADP - and NAD + ME- type C plants and contributes to C will focus on the functional aspects of PPDK in 4 4 photosynthetic PEP supply in PEPcK-type C4 the C4 pathway and the more recent findings con- cerning its post-translational regulation. plants (Ashton et al., 1990 ) . A. Role of PPDK in C Plants 4 B. PPDK Enzyme Properties In the C pathway, PPDK catalyzes the conversion 4 1. Catalysis as Related to Structure of 3-carbon pyruvate into phospho enol pyruvate Most of what is known concerning the structural aspects of the PPDK catalytic mechanism origi- Abbreviations : aa – amino acid; GFP – green fluorescent nate from studies of crystallized PPDK homo- protein; NADP MDH – NADP malate dehydrogenase; NADP dimer from the bacterium Clostridium symbiosum ME – NADP malic enzyme; ORF – open reading frame; Pi – (Pocalyko et al., 1990 ; Herzberg et al., 1996 ; Lin inorganic phosphate; PPi – pyrophosphate; PEP – phospho - enol pyruvate; PEPc – PEP carboxylase; PPD K – pyruvate et al., 2006 ; Lim et al., 2007 ) . Although compara- orthophosphate dikinase; Pyr – pyruvate; RP – regulatory ble studies of crystallized plant PPDK are not as protein; yet available, the C. symbiosum structural model 303 15 C4 PPDK and C4 PPDK regulatory protein is considered to be homologous to that of the plant et al., 1996 ; Lin et al., 2006 ; Lim et al., 2007 ) . enzyme as indicted by a high degree of conserved A key element in this mechanism is the ability primary structure between plant and bacterial of the central domain to freely pivot or swivel PPDKs (Pocalyko et al., 1990 ) , and an identical between the remote N- and C-terminal domains reaction mechanism (Carroll et al., 1990 ) . Fur- upon flanking “hinge-like” peptide linkers. Thus, thermore, the first reported plant PPDK crystal as viewed in this structural context, catalysis structure (of a maize C4 PPDK dimer complexed proceeds within these domains through a 3-step with PEP) is very similar to the three dimensional partial reaction sequence as illustrated in Fig. 1 . structure of the C. symbiosum enzyme (Nakanishi In the C4 PEP-forming direction, the first par- et al., 2005 ) . tial reaction is initiated by ATP binding to the PPDK is a member of the PEP-utilizing enzyme N-terminal nucleotide-binding domain. This is