Discovery of Novel Phosphoenolpyruvate Carboxylase (PEPC) Genes and Their Active Polypeptides in the Green Microalga Chlamydomonas reinhardtii

This work describes the discovery of novel phosphoenolpyruvate carboxylase (PEPC) genes and their
active catalitic polypeptides in the green microalga Chlamydomonas reinhardhii. Green-algal PEPC has
been unexplored before in molecular terms. Our recent studies have reported the molecular cloning of
the two PEPC genes, Ppc genes in Creinhardtii (CrPpcl, CrPpc2), each of which is transcribed in vivo
and encodes a fully active, recombinant PEPC that lacks the regulatory, N-terminal seryl-
phosphorylation domain typifying the vascular-plant enzyme. These distinct catalytic subunit-types
differ with respect to their (a) predicted molecular mass (-1889 (C-Ppcl) versus -131.2
and critical C-terminal tetrapeptide; and (b) immuno-reactivity with antisera against the p102 and
p130 polypeptides of Sminutum PEPCI/PEPC2 and PE and PEPC2. respectively. Only the Ppel transcript
encodes the p102 catalytic subunits common to both to both Class-1 and Class-2 enzyme-forms in
Creinhard. We studied We studied the distribution of these two encoded catalytic subunits in the minor C Class-1
and predominant Class-2 PEPC enzyme-forms, the latter of which is a novel high-molecular-mass, he
tero-oligomeric complex containing both Opel (p109) and CP2 (p131) pobpeptides. The Class-1
enzyme, however, is a typical PEPC homotetramer comprised solely of p108. The steady-state tran-
script levels of both Opel2 are coordinatch up-down-regulated by changes in [CO] or [NL] dar
ing growth, and generally mirror the response of cytoplasmic glutamine synthetase (G) transcript
abundance to changes in inorganic [N] at 5% CO We also documented that the amount of both
CPpel/2 catalytic subunits is up-down-regulated by varying levels of NIL supplied to the culture
medium. To our knowledge, these collective findings provide the first molecular insight into the Pe
genes and corresponding PEPC catalytic subunits in any cukaryotic alga