Maize leaf phosphoenolpyruvate carboxylase: phosphorylation of Ser15 with a mammalian cyclic AMP-dependent protein kinase diminishes sensitivity to inhibition by malate

AbstractThe so-called light-activation of phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31) involved in C4 photosynthesis is known to be mediated by phosphorylation. A cyclic AMP-dependent protein kinase from bovine heart was found to be able to phosphorylate PEPC. The phosphorylation was accompanied by the changes in kinetic properties, which were very similar to the reported light activation. The phosphorylated amino acid residue was identified as Ser and the position of this Ser on the primary structure [(1988) FEBS Lett. 229, 107-110] was determined to be Ser15

Plausible phosphoenolpyruvate binding site revealed by 2.6 Å structure of Mn2+-bound phosphoenolpyruvate carboxylase from Escherichia coli11The coordinates and structure factors have been deposited in the Protein Data Bank (accession number 1QB4).

AbstractWe have determined the crystal structure of Mn2+-bound Escherichia coli phosphoenolpyruvate carboxylase (PEPC) using X-ray diffraction at 2.6 Å resolution, and specified the location of enzyme-bound Mn2+, which is essential for catalytic activity. The electron density map reveals that Mn2+ is bound to the side chain oxygens of Glu-506 and Asp-543, and located at the top of the α/β barrel in PEPC. The coordination sphere of Mn2+ observed in E. coli PEPC is similar to that of Mn2+ found in the pyruvate kinase structure. The model study of Mn2+-bound PEPC complexed with phosphoenolpyruvate (PEP) reveals that the side chains of Arg-396, Arg-581 and Arg-713 could interact with PEP

Transcriptome Profiling of Lotus japonicus Roots During Arbuscular Mycorrhiza Development and Comparison with that of Nodulation

To better understand the molecular responses of plants to arbuscular mycorrhizal (AM) fungi, we analyzed the differential gene expression patterns of Lotus japonicus, a model legume, with the aid of a large-scale cDNA macroarray. Experiments were carried out considering the effects of contaminating microorganisms in the soil inoculants. When the colonization by AM fungi, i.e. Glomus mosseae and Gigaspora margarita, was well established, four cysteine protease genes were induced. In situ hybridization revealed that these cysteine protease genes were specifically expressed in arbuscule-containing inner cortical cells of AM roots. On the other hand, phenylpropanoid biosynthesis-related genes for phenylalanine ammonia-lyase (PAL), chalcone synthase, etc. were repressed in the later stage, although they were moderately up-regulated on the initial association with the AM fungus. Real-time RT–PCR experiments supported the array experiments. To further confirm the characteristic expression, a PAL promoter was fused with a reporter gene and introduced into L. japonicus, and then the transformants were grown with a commercial inoculum of G. mosseae. The reporter activity was augmented throughout the roots due to the presence of contaminating microorganisms in the inoculum. Interestingly, G. mosseae only colonized where the reporter activity was low. Comparison of the transcriptome profiles of AM roots and nitrogen-fixing root nodules formed with Mesorhizobium loti indicated that the PAL genes and other phenylpropanoid biosynthesis-related genes were similarly repressed in the two organs

Interplay of light and temperature during the in planta modulation of C4 phosphoenolpyruvate carboxylase from the leaves of Amaranthus hypochondriacus L.: diurnal and seasonal effects manifested at molecular levels

The interactive effects of light and temperature on C4 phosphoenolpyruvate carboxylase (PEPC) were examined both in vivo and in situ using the leaves of Amaranthus hypochondriacus collected at different times during a day and in each month during the year. The maximum activity of PEPC, least inhibition by malate, and highest activation by glucose-6-phosphate were at 15.00 h during a typical day, in all the months. This peak was preceded by maximum ambient light but coincided with high temperature in the field. The highest magnitude in such responses was in the summer (e.g. May) and least in the winter (e.g. December). Light appeared to dominate in modulating the PEPC catalytic activity, whereas temperature had a strong influence on the regulatory properties, suggesting interesting molecular interactions. The molecular mechanisms involved in such interactive effects were determined by examining the PEPC protein/phosphorylation/mRNA levels. A marked diurnal rhythm could be seen in the PEPC protein levels and phosphorylation status during May (summer month). In contrast, only the phosphorylation status increased during the day in December (winter month). The mRNA peaks were not as strong as those of phosphorylation. Thus, the phosphorylation status and the protein levels of PEPC were crucial in modulating the daily and seasonal patterns in C4 leaves in situ. This is the first detailed study on the diurnal as well as seasonal patterns in PEPC activity, its regulatory properties, protein levels, phosphorylation status, and mRNA levels, in relation to light and temperature intensities in the field

A plastidial sodium-dependent pyruvate transporter (vol 476, pg 472, 2011)

Furumoto T, Yamaguchi T, Ohshima-Ichie Y, et al. A plastidial sodium-dependent pyruvate transporter (vol 476, pg 472, 2011). Nature. 2011;478(7368):274

ダイチョウキン フォスフォエノルピルビンサン カルボキシラーゼ ニ オケル アロステリック コウカ ニ カンスル ケンキュウ

京都大学0048新制・論文博士博士(理学)乙第2234号論理博第422号新制||理||175(附属図書館)UT51-48-E584(主査)教授 香月 裕彦, 教授 大杉 治郎, 教授 波多野 博行, 教授 由良 隆学位規則第5条第2項該当Doctor of ScienceKyoto UniversityDFA

Regulatory phosphorylation of plant phosphoenolpyruvate carboxylase: role of a conserved basic residue upstream of the phosphorylation site

AbstractIn order to mimic regulatory phosphorylation of the Ser-15 of maize C4-form phosphoenolpyruvate carboxylase (PEPC), we replaced Ser-15 and Lys-12 with Asp (S15D) and Asn (K12N), respectively, by site-directed mutagenesis. Although both mutant enzymes were catalytically as active as the wild-type PEPC, they showed much less sensitivity to malate, an allosteric inhibitor, similarly to the phosphorylated wild-type PEPC. A maize protein kinase of 30 kDa which is known to be specific to PEPC (PEPC-PK), phosphorylated K12N as well as the wild-type PEPC but not S15D. The phosphorylation of K12N further diminished the sensitivity to malate. Thus, a positive charge of the conserved Lys-12 is not required for the recognition by PEPC-PK but contributes to the intrinsic sensitivity to malate inhibition