Identification and expression analysis of two inorganic C- and N-responsive genes encoding novel and distinct molecular forms of eukaryotic phosphoenolpyruvate carboxylase in the green microalga Chlamydomonas reinhardtii.
Phosphoenolpyruvate carboxylase (PEPC [Ppc]) has been previously purified and characterized in biochemical and immunological terms from two green microalgae, Chlamydomonas reinhardtii and Selenastrum minutum. The findings indicate that these algae possess at least two distinct PEPC enzyme-forms, homotetrameric Class-1 and heteromeric Class-2, that differ significantly from each other and their plant and prokaryotic counterparts. Surprisingly, however, green-algal PEPC has been unexplored to date in molecular terms. This study reports the molecular cloning of the two Ppc genes in C. reinhardtii (CrPpc1, CrPpc2), each of which is transcribed in vivo and encodes a fully active, recombinant PEPC that lacks the regulatory, N-terminal seryl-phosphorylation domain that typifies the vascular-plant enzyme. These distinct catalytic subunit-types differ with respect to their (i) predicted molecular mass ( approximately 108.9 [CrPpc1] versus approximately 131.2 kDa [CrPpc2]) and critical C-terminal tetrapeptide; and (ii) immunoreactivity with antisera against the p102 and p130 polypeptides of S. minutum PEPC1/PEPC2 and PEPC2, respectively. Only the Ppc1 transcript encodes the p102 catalytic subunits common to both Class-1 and Class-2 enzyme-forms in C. reinhardtii. The steady-state transcript levels of both CrPpc1/2 are coordinately up-/down-regulated by changes in [CO2] or [NH] during growth, and generally mirror the response of cytoplasmic glutamine synthetase (Gs1) transcript abundance to changes in inorganic [N] at 5% CO2. These collective findings provide key molecular insight into the Ppc genes and corresponding PEPC catalytic subunits in the eukaryotic algae.
Mamedov
Tarlan G
TG
Department of Biochemistry, University of Nebraska-Lincoln, George W. Beadle Center, Lincoln, NE 68588-0664, USA.
Moellering
Eric R
ER
Chollet
Raymond
R
D000595:Amino Acid Sequence; D000818:Animals; D002244:Carbon / Q000502:physiology*; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics*; D007527:Isoenzymes; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000502:physiology*; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics*; D010802:Phylogeny; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D007527:Isoenzymes; D002244:Carbon; D010730:Phosphoenolpyruvate Carboxylase; D009584:Nitrogen