Pyruvate Decarboxylase from Pisum sativum

Abstract

To study the molecular structure and function of pyruvate decarboxylase (PDC) from plants the protein was isolated from pea seeds and partially characterised. The active enzyme which occurs in the form of higher oligomers consists of two different subunits appearing in SDSiPAGE and mass spectroscopy experiments. For further experiments, like X-ray crystallography, it was necessary to elucidate the protein sequence.Partial cDNA clones encoding pyruvate decarboxylase from seeds of Pisum sativum cv. Miko have been obtained by means of polymerase chain reaction techniques. The first sequences were found using degenerate oligonucleotide primers designated according to conserved amino acid sequences of known pyruvate decarboxylases. The missing parts of one cDNA were amplified applying the 3'-and 5'-rapid amplification of cDNA ends systems. The amino acid sequence deduced from the entire cDNA sequence displays strong similarity to pyruvate decarboxylases from other organisms, especially from plants. A molecular mass of 64 kDa was calculated for this protein correlating with estimations for the smaller subunit of the oligomeric enzyme. The PCR experiments led to at least three different clones representing the middle part of the PDC cDNA indicating the existence of three isozymes. Two of these isoforms could be confirmed on the protein level by sequencing tryptic peptides. Only anaerobically treated roots showed a positive signal for PDC mRNA in Northern analysis although the cDNA from imbibed seeds was successfully used for PCR.

Small-Angle X-ray Solution-Scattering Studies on Ligand-Induced Subunit Interactions of the Thiamine Diphosphate Dependent Enzyme Pyruvate Decarboxylase from Different Organisms

“…As already mentioned, PDCs from plant seeds (e.g., maize, beans, wheat) have generally more complex oligomeric structures. Interestingly though, the values of the kinetic parameters such as substrate affinity, catalytic activity, and reconstitution rate with the cofactors are close to those of the yeast enzyme (10,14,15). Recent studies of enzyme activation and thiol group modifications have, however, revealed significant quantitative differences (10), and the SAXS experiments were aimed at correlating these with possible structural differences.…”

“…This enzyme has a lower K M for its substrate pyruvate (0.4 mM (13) compared to 1 mM for the activated state of the yeast and plant enzyme, respectively (9,14)) and higher catalytic activity (180 U/mg compared to 60-80 U/mg for the others). The catalytically active quaternary forms are tetramers in yeast and bacterial PDC and higher oligomers in the enzyme from germinating pea seeds (14) as well as in other plant species (15); however, there is no precise description of the catalytically active states of plant PDCs.…”

Enhanced Low Oxygen Survival in Arabidopsis through Increased Metabolic Flux in the Fermentative Pathway

“…4). Biochemical characterizations have demonstrated that, like yeast and some bacterial PDC enzymes, plant PDC has nonlinear kinetics and is activated by its substrate pyruvate (Mü cke et al, 1996;Dietrich and Kö nig, 1997). The PDC2 enzyme, which is constitutively expressed at low levels in roots, is quite different from the anaerobically induced enzyme (R. Dolferus, unpublished data) and may have different kinetic properties.…”

Regulation of alcoholic fermentation in batch and chemostat cultures ofKluyveromyces lactis CBS 2359

“…Preliminary transcriptional analysis has revealed that in K. lactis, this regulation occurs at least partly at the level of transcription of the KIPDC1 gene (A. Zeeman, unpublished). Regulation of pyruvate decarboxylase synthesis by oxygen also occurs in plants (Mü cke et al, 1996). In view of its genetic accessibility and the presence of a single structural gene encoding pyruvate decarboxylase (Bianchi et al, 1996), K. lactis is an interesting laboratory system to study regulation of PDC gene expression by oxygen in yeast and to compare this with plant systems.…”