PURIFICATION OF THE PYRUVATE-DEHYDROGENASE MULTIENZYME COMPLEX OF ZYMOMONAS-MOBILIS AND IDENTIFICATION AND SEQUENCE-ANALYSIS OF THE CORRESPONDING GENES

The pyruvate dehydrogenase (PDH) complex of the gram-negative bacteriu m Zymomonas mobilis was purified to homogeneity. From 250 g of cells, we isolated 1 mg of PDH complex with a specific activity of 12.6 U/mg of protein, Analysis of subunit composition revealed a PDH (E1) consis ting of the two subunits E1 alpha (38 kDa) and E1 beta (56 kDa), a dih ydrolipoamide acetyltransferase (E2) of 48 kDa, and a lipoamide dehydr ogenase (E3) of 50 kDa, The E2 core of the complex is arranged to form a pentagonal dodecahedron, as shown by electron microscopic images, r esembling the quaternary structures of PDH complexes from gram-positiv e bacteria and eukaryotes. The PDH complex-encoding genes were identif ied by hybridization experiments and sequencer analysis in two separat e gene regions in the genome of Z. mobilis. The genes pdhA alpha (1,06 5 bp) and pdhA beta (1,389 bp), encoding the E1 alpha and E1 beta subu nits of the E1 component, were located downstream of the gene encoding enolase, The pdhB (1,323 bp) and lpd (1,401 bp) genes, encoding the E 2 and E3 components, were identified in an unrelated gene region toget her with a 450-bp open reading frame (ORF) of unknown function in the order pdhB-ORF2-lpd. Highest similarities of the gene products of the pdhA alpha, pdhA beta, and pdhB genes were found with the correspondin g enzymes of Saccharomyces cerevisiae and other eukaryotes. Like the d ihydrolipoamide acetyltransferases of S. cerevisiae and numerous other organisms, the product of the pdhB gene contains a single lipoyl doma in, The E1 beta subunit PDH was found to contain an amino-terminal lip oyl domain, a property which is unique among PDHs.