Improved purification of pyruvate decarboxylase from wheat germ

Abstract

An improved procedure was developed for the isolation of pyruvate decarboxylase from wheat germ. Its final step, an electrophoresis of the native apoenzyme in concave pore gradient polyacrylamide gels, followed by superficial activity-staining, produced two bands of different molecular masses and chain compositions. The highmolecular-mass band occurred in low quantity and consisted of, probably eight, apparently identical chains of M , = 33 000, as judged from sodium dodecyl sulfate electrophoreses. The low-molecular-mass band contained two types of chains with MF = 63000-65000 and M! = 61000-62000. The N termini of both chains were threonine, whereas their C-terminal sequences were different:Their amino acid composition was too different to be compatible with our original concept of one chain being produced from the other by proteolytic shortening. Limited proteolysis by Staphylococcus aureus V8 proteinase yielded peptides partly identical size and partly quite different.In all properties investigated, the low-molecular-mass enzyme largely resembled yeast pyruvate decarboxylase; the holoenzyme appeared to possess ( c I~)~ structure, the apoenzyme a/?. SH reagents inactivated the enzyme. Binding and fluorescence of 2-p-toluidinonaphthalene-6-sulfonate indicated a similar lipophilicity of the active site as found earlier for the yeast enzyme. 2-Hydroxy-5-nitrobenzyl modification of exposed tryptophan residues left the holoenzyme intact, but in the apoenzyme it destroyed most of the cofactor-binding ability and hence the activity. The strength of cofactor binding and the maximal specific activity were found somewhat lower than in yeast pyruvate decarboxylase.Pyruvate decarboxylase (PDC) is known to occur in yeasts, several other fungi, many plant materials (review: [l]) and a few bacteria [2, 31. One of the early sources for its preparation has been wheat germ [4]. When our efforts to achieve a detailed chemical analysis of yeast PDC became hampered by uncontrollable actions of contaminating proteinases [5], a practical alternative seemed to be the investigation of PDC from wheat germ, which was expected to contain fewer proteinases. The original method for the preparation of wheat germ PDC [4] has been applied without major alterations in all instances when this enzyme was hitherto needed for a variety of purposes [6 -111. Re-examination of this procedure with modern electrophoresis methods demonstrated that it yields PDC of insufficient chain homogeneity for the requirements of peptide mapping and sequence analysis. Therefore a more efficient isolation scheme was elaborated.

Production of the Gram-positive Sarcina ventriculi pyruvate decarboxylase in Escherichia coli The GenBank accession number for the sequence reported in this paper is AF354297.

“…The high-affinity K m value of 2n8 mM obtained for the S. ventriculi PDC is similar to the K m values obtained with many fungal and plant PDCs, including those purified from Saccharomyces cerevisiae (1-3 mM) (Boiteux & Hess, 1970 ;Hu$ bner et al, 1978), Zygosaccharomyces bisporus (1n73 mM) (Neuser et al, 2000), orange (0n8-3n2 mM) (Raymond et al, 1979), and wheat germ (3 mM) (Zehender et al, 1987). However, these K m values are several-fold higher than those reported for the PDCs of Z. mobilis (0n3-0n4 mM) (Bringer-Meyer et al, 1986 ;Neale et al, 1987b) and rice (0n25 mM) (Rivoal et al, 1990).…”

Pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon, Pyrococcus furiosus , functions as a CoA-dependent pyruvate decarboxylase

“…The decarboxylation reaction of POR must therefore differ from that of pyruvate decarboxylases. Pyruvate decarboxylases have been purified from a variety of organisms (37)(38)(39)(40)(41) and the crystal structure of one is known (42,43). That they are also TPP-containing enzymes and the mechanism of pyruvate decarboxylation, which does involve direct conversion of the hydroxyethyl-TPP complex to the aldehyde, is well understood (43)(44)(45).…”

Maize pyruvate decarboxylase mRNA is induced anaerobically

“…They also found that the M r 44 000 polypeptide increased in amount during storage. Zehender et al [34] found a major form of wheat germ PDC to have two type of chains: alpha of 63 000-65 000, and beta of61000-62000. Laszlo [20] provided data suggesting that the smaller subunit was derived from the larger subunit by proteolytic cleavage.…”