Purification of the Pyruvate Dehydrogenase Multienzyme Complex of Zymomonas mobilis and Identification and Sequence Analysis of the Corresponding Genes

Abstract

The pyruvate dehydrogenase (PDH) complex of the gram-negative bacterium 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) consisting of the two subunits E1α (38 kDa) and E1β (56 kDa), a dihydrolipoamide acetyltransferase (E2) of 48 kDa, and a lipoamide dehydrogenase (E3) of 50 kDa. The E2 core of the complex is arranged to form a pentagonal dodecahedron, as shown by electron microscopic images, resembling the quaternary structures of PDH complexes from gram-positive bacteria and eukaryotes. The PDH complex-encoding genes were identified by hybridization experiments and sequence analysis in two separate gene regions in the genome ofZ. mobilis. The genes pdhAα (1,065 bp) andpdhAβ (1,389 bp), encoding the E1α and E1β subunits 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 E2 and E3 components, were identified in an unrelated gene region together 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 pdhAα,pdhAβ, and pdhB genes were found with the corresponding enzymes of Saccharomyces cerevisiae and other eukaryotes. Like the dihydrolipoamide acetyltransferases of S. cerevisiae and numerous other organisms, the product of thepdhB gene contains a single lipoyl domain. The E1β subunit PDH was found to contain an amino-terminal lipoyl domain, a property which is unique among PDHs.

Exceptional characteristics of heterotetrameric (α2β2) E1p of the pyruvate dehydrogenase complex from Zymomonas mobilis: expression from an own promoter and a lipoyl domain in E1β

“…In strain ZM4 the lpd gene was mapped by hybridization more than 800 kb downstream of adhB (encoding alcohol dehydrogenase II); adhB is located close to pdhAL ( [11], embl: AF086791). This is in good agreement with our earlier results that the structural genes (pdhAK, pdhAL, pdhB and lpd) encoding the PDHC of Z. mobilis ATCC 29191 were located in two distinct chromosomal regions organized in a pdhAK-pdhAL gene cluster and a pdhB-orf2lpd gene cluster [3,7]. In this work the presence of three promoters for expression of the pdh genes of Z. mobilis is shown.…”

“…In contrast, P lpd is negatively regulated by ArcA [10]. The Z. mobilis strain ATCC 29191 used in this study has been shown to have two separate pdh gene clusters (pdhAK-pdhAL and pdhB-orf2-lpd) [3]. Previously the pdhKL region of Z. mobilis ZM4 ( [11], embl: AF086791) was sequenced and mapped, revealing more than 96% identity to the nucleotide sequence of the pdhAKL region of Z. mobilis ATCC 29191 [3].…”

“…Lipoyl domains are commonly found in the core-forming transacetylase protein (E2p) and in the protein X components of eukaryotic PDHC [2]. In Zymomonas mobilis the Nterminal part of E2 contains a single lipoyl domain, followed by an E1 and/or E3 subunit binding domain and a C-terminal acetyltransferase active site, which also forms the structural dodecahedral core of the complex [3]. The domains are connected by linker segments rich in charged amino acid residues.…”

“…The £exibility of the linker segments is essential for this active site coupling [6]. In Z. mobilis an additional lipoyl domain has been located at the N-terminus of the L subunit of the heterotetrameric (K 2 L 2 ) E1p [3]. It is connected by a linker segment to the C-terminal part of this subunit as illustrated in Fig.…”

“…The Z. mobilis strain ATCC 29191 used in this study has been shown to have two separate pdh gene clusters (pdhAK-pdhAL and pdhB-orf2-lpd) [3]. Previously the pdhKL region of Z. mobilis ZM4 ( [11], embl: AF086791) was sequenced and mapped, revealing more than 96% identity to the nucleotide sequence of the pdhAKL region of Z. mobilis ATCC 29191 [3]. In strain ZM4 the lpd gene was mapped by hybridization more than 800 kb downstream of adhB (encoding alcohol dehydrogenase II); adhB is located close to pdhAL ( [11], embl: AF086791).…”