Diversity in guanosine 3',5'-bisdiphosphate (ppGpp) sensitivity among guanylate kinases of bacteria and plants.
The guanosine 3',5'-bisdiphosphate (ppGpp) signaling system is shared by bacteria and plant chloroplasts, but its role in plants has remained unclear. Here we show that guanylate kinase (GK), a key enzyme in guanine nucleotide biosynthesis that catalyzes the conversion of GMP to GDP, is a target of regulation by ppGpp in chloroplasts of rice, pea, and Arabidopsis. Plants have two distinct types of GK that are localized to organelles (GKpm) or to the cytosol (GKc), with both enzymes being essential for growth and development. We found that the activity of rice GKpm in vitro was inhibited by ppGpp with a Ki of 2.8 μM relative to the substrate GMP, whereas the Km of this enzyme for GMP was 73 μM. The IC50 of ppGpp for GKpm was ∼10 μM. In contrast, the activity of rice GKc was insensitive to ppGpp, as was that of GK from bakers' yeast, which is also a cytosolic enzyme. These observations suggest that ppGpp plays a pivotal role in the regulation of GTP biosynthesis in chloroplasts through specific inhibition of GKpm activity, with the regulation of GTP biosynthesis in chloroplasts thus being independent of that in the cytosol. We also found that GKs of Escherichia coli and Synechococcus elongatus PCC 7942 are insensitive to ppGpp, in contrast to the ppGpp sensitivity of the Bacillus subtilis enzyme. Our biochemical characterization of GK enzymes has thus revealed a novel target of ppGpp in chloroplasts and has uncovered diversity among bacterial GKs with regard to regulation by ppGpp.
From the Graduate School of Science and Engineering, Ehime University, Matsuyama 790-8577, Japan, the Proteo-Science Center and the Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan.
Yuhta
Y
Nomura
the Proteo-Science Center and the Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan.
Atsushi
A
Izumi
From the Graduate School of Science and Engineering, Ehime University, Matsuyama 790-8577, Japan, the Proteo-Science Center and the Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan.
Yoshinori
Y
Fukunaga
the Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka 812-8581, Japan.
Kensuke
K
Kusumi
the Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka 812-8581, Japan.
Koh
K
Iba
the Faculty of Agriculture, Ehime University, Matsuyama 790-8566, Japan.
Seiya
S
Watanabe
the Proteo-Science Center and the Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan.
Yoichi
Y
Nakahira
the Institute for Plant Biochemistry, Cluster of Excellence on Plant Sciences, Heinrich-Heine University, Düsseldorf D-40225, Germany.
Andreas P M
AP
Weber
the Proteo-Science Center and the Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan.
Akira
A
Nozawa
the Proteo-Science Center and the Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan, tozawa.yuzuru.mx@ehime-u.ac.jp.
Yuzuru
Y
Tozawa
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D014644:Genetic Variation; D006159:Guanosine Tetraphosphate / Q000378:metabolism*; D051528:Guanylate Kinases / Q000235:genetics / Q000378:metabolism*; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology / Q000235:genetics; D018532:Pisum sativum / Q000201:enzymology / Q000235:genetics; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology; D046940:Synechococcus / Q000201:enzymology / Q000235:genetics
D006159:Guanosine Tetraphosphate; D051528:Guanylate Kinases; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases
Bacteria; Chloroplast; Enzymes; Guanylate Kinase; Metabolism; Nucleoside Nucleotide Biosynthesis; Oryza Sativa; Plant; Stringent Response; ppGpp