MOLECULAR-BIOLOGY OF C-4 PHOSPHOENOLPYRUVATE CARBOXYLASE - STRUCTURE,REGULATION AND GENETIC-ENGINEERING

Three to four families of nuclear genes encode different isoforms of p hosphoenolpyruvate (PEP) carboxylase (PEPC): C-4-specific, C-3 or etio lated, CAM and root forms. C-4 leaf PEPC is encoded by a single gene ( ppc) in sorghum and maize, but multiple genes in the C-4-dicot Flaveri a trinervia. Selective expression of ppc in only C-4-mesophyll cells i s proposed to be due to nuclear factors, DNA methylation and a distinc t gene promoter. Deduced amino acid sequences of C-4-PEPC pinpoint the phosphorylatable serine near the N-terminus, C-4-specific valine and serine residues near the C-terminus, conserved cysteine, lysine and hi stidine residues and PEP binding/catalytic sites. During the PEPC reac tion, PEP and bicarbonate are first converted into carboxyphosphate an d the enolate of pyruvate. Carboxyphosphate decomposes within the acti ve site into Pi and CO2, the latter combining with the enolate to form oxalacetate. Besides carboxylation, PEPC catalyzes a HCO3-dependent h ydrolysis of PEP to yield pyruvate and Pi. Post-translational regulati on of PEPC occurs by a phosphorylation/dephosphorylation cascade in vi vo and by reversible enzyme oligomerization in vitro. The interrelatio n between phosphorylation and oligomerization of the enzyme is not cle ar. PEPC-protein kinase (PEPC-PK), the enzyme responsible for phosphor ylation of PEPC, has been studied extensively while only limited infor mation is available on the protein phosphatase 2A capable of dephospho rylating PEPC. The C-4 ppc was cloned and expressed in Escherichia coi l as well as tobacco. The transformed E. coli produced a functional/ph osphorylatable C-4 PEPC and the transgenic tobacco plants expressed bo th C-3 and C-4 isoforms. Site-directed mutagenesis of ppc indicates th e importance of His(138), His(579) and Arg(587) in catalysis and/or su bstrate-binding by the E. coli enzyme, Ser(8) in the regulation of sor ghum PEPC. Important areas for further research on C-4 PEPC are: mecha nism of transduction of light signal during photoactivation of PEPC-PK and PEPC in leaves, extensive use of site-directed mutagenesis to pre cisely identify other key amino acid residues, changes in quarternary structure of PEPC in vivo, a high-resolution crystal structure, and ho rmonal regulation of PEPC expression.