Biochemical and molecular characterization of the Clostridium magnum acetoin dehydrogenase enzyme system

Abstract

E2 (dihydrolipoamide acetyltransferase) and E3 (dihydrolipoamide dehydrogenase) of the Clostridium magnum acetoin dehydrogenase enzyme system were copurified in a three-step procedure from acetoin-grown cells. The denatured E2-E3 preparation comprised two polypeptides with M(r)s of 49,000 and 67,000, respectively. Microsequencing of both proteins revealed identical amino acid sequences. By use of oligonucleotide probes based on the N-terminal sequences of the alpha and beta subunits of E1 (acetoin dehydrogenase, thymine PPi dependent), which were purified recently (H. Lorenzl, F.B. Oppermann, B. Schmidt, and A. Steinbüchel, Antonie van Leeuwenhoek 63:219-225, 1993), and of E2-E3, structural genes acoA (encoding E1 alpha), acoB (encoding E1 beta), acoC (encoding E2), and acoL (encoding E3) were identified on a single ClaI restriction fragment and expressed in Escherichia coli. The nucleotide sequences of acoA (978 bp), acoB (999 bp), acoC (1,332 bp), and acoL (1,734 bp), as well as those of acoX (996 bp) and acoR (1,956 bp), were determined. The amino acid sequences deduced from acoA, acoB, acoC, and acoL for E1 alpha (M(r), 35,532), E1 beta (M(r), 35,541), E2 (M(r), 48,149), and E3 (M(r), 61,255) exhibited striking similarities to the amino acid sequences of the corresponding components of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system and the Alcaligenes eutrophus acetoin-cleaving system, respectively. Significant homologies to the enzyme components of various 2-oxo acid dehydrogenase complexes were also found, indicating a close relationship between the two enzyme systems. As a result of the partial repetition of the 5' coding region of acoC into the corresponding part of acoL, the E3 component of the C. magnum acetoin dehydrogenase enzyme system contains an N-terminal lipoyl domain, which is unique among dihydrolipoamide dehydrogenases. We found strong similarities between the AcoR and AcoX sequences and the A. eutrophus acoR gene product, which is a regulatory protein required for expression of the A. eutrophus aco genes, and the A. eutrophus acoX gene product, which has an unknown function, respectively. The aco genes of C. magnum are probably organized in one single operon (acoABXCL); acoR maps upstream of this operon.

Molecular structure of the lipoamide dehydrogenase domain of a surface antigen from Neisseria meningitidis

“…It is interesting to note that the p64k is one of the rare dihydrolipoamide dehydrogenases containing an aminoterminal lipoyl domain (Silva et al, 1992). Three other enzymes with equivalent structure have been reported from Alcaligenes eutrophus (Hein & Steinbuchel, 1994), Clostridium magnum (Kruger et al, 1994) and Mycoplasma capricolum (Zhu & Peterkofsky, 1996). It may be possible that the oxidative reaction proceeds in the complex either through the association of E2 and E3 (the usual known process) or via this dihydrolipoamide dehydrogenase containing a lipoyl domain, as discussed by Zhu & Peterkofsky, (1996).…”

Enzymatic Characterization of Dihydrolipoamide Dehydrogenase from Streptococcus pneumoniae Harboring Its Own Substrate

“…The sequence encoding the lipoyl protein domain is no longer part of the acetyltransferase gene, but has moved to become the N-terminal portion of the downstream dldh gene. S. pneumoniae shares this property with other Streptococcus and Clostridium species (16,38,39). DLDH in some Mycoplasma species as well as in N. meningitidis (40,41) also contains an N-terminal lipoyl protein domain.…”

Novel esterase activity of dihydrolipoamide acetyltransferase AcoC of Pseudomonas putida identified by mutation of the acoR regulator

“…Regulatory genes (designated acoR or acoK) for the respective aco operons have been identified in A. eutrophus, C. magnum, and K. pneumoniae (17)(18)(19). In P. putida an acoR-like gene has not yet been identified.…”