Molecular biology of C phospho enolpyruvate carboxylase: Structure, regulation and genetic engineering

Three to four families of nuclear genes encode different isoforms of phospho enolpyruvate (PEP) carboxylase (PEPC): C-specific, C or etiolated, CAM and root forms. C leaf PEPC is encoded by a single gene (ppc) in sorghum and maize, but multiple genes in the C-dicot Flaveria trinervia. Selective expression of ppc in only C-mesophyll cells is proposed to be due to nuclear factors, DNA methylation and a distinct gene promoter. Deduced amino acid sequences of C-PEPC pinpoint the phosphorylatable serine near the N-terminus, C-specific valine and serine residues near the C-terminus, conserved cysteine, lysine and histidine residues and PEP binding/catalytic sites. During the PEPC reaction, PEP and bicarbonate are first converted into carboxyphosphate and the enolate of pyruvate. Carboxyphosphate decomposes within the active site into Pi and CO, the latter combining with the enolate to form oxalacetate. Besides carboxylation, PEPC catalyzes a HCO-dependent hydrolysis of PEP to yield pyruvate and Pi. Post-translational regulation of PEPC occurs by a phosphorylation/dephosphorylation cascade in vivo and by reversible enzyme oligomerization in vitro. The interrelation between phosphorylation and oligomerization of the enzyme is not clear. PEPC-protein kinase (PEPC-PK), the enzyme responsible for phosphorylation of PEPC, has been studied extensively while only limited information is available on the protein phosphatase 2A capable of dephosphorylating PEPC. The Cppc was cloned and expressed in Escherichia coli as well as tobacco. The transformed E. coli produced a functional/phosphorylatable C PEPC and the transgenic tobacco plants expressed both C and C isoforms. Site-directed mutagenesis of ppc indicates the importance of His, His and Arg in catalysis and/or substrate-binding by the E. coli enzyme, Ser in the regulation of sorghum PEPC. Important areas for further research on C PEPC are: mechanism of transduction of light signal during photoactivation of PEPC-PK and PEPC in leaves, extensive use of site-directed mutagenesis to precisely identify other key amino acid residues, changes in quarternary structure of PEPC in vivo, a high-resolution crystal structure, and hormonal regulation of PEPC expression. © 1994 Kluwer Academic Publishers.