Expression of the sodium ion pump methylmalonyl-coenzyme A-decarboxylase from Veillonella parvula and of mutated enzyme specimens in Escherichia coli

Abstract

The structural genes of the sodium ion pump methylmalonyl-coenzyme A (CoA)-decarboxylase from Veillonella parvula have recently been cloned on three overlapping plasmids (pJH1, pJH20, and pJH40) and sequenced. To synthesize the complete decarboxylase in Escherichia coli, the genes were fused in the correct order (mmdADECB) on a single plasmid (pJH70). A DNA region upstream of mmdA apparently served as promoter in E. coli because expression of the mmd genes was not dependent on the correct orientation of the lac promoter present on the pBluescript KS؉-derived expression plasmid. To allow controlled induction of the mmd genes, the upstream region was deleted and the mmd genes were cloned behind a T7 promoter. The derived plasmid, pT7mmd, was transformed into E. coli BL21(DE3) expressing T7 RNA polymerase under the control of the lac promoter. The synthesized proteins showed the typical properties of methylmalonyl-CoAdecarboxylase, i.e., the same migration behavior during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, stimulation of the decarboxylation activity by sodium ions, and inhibition with avidin. In methylmalonyl-CoA-decarboxylase expressed in E. coli from pT7mmd, the ␥ subunit was only partially biotinylated and the ␣ subunit was present in substoichiometric amounts, resulting in a low catalytic activity. This activity could be considerably increased by coexpression of biotin ligase and by incubation with separately expressed ␣ subunit. After these treatments methylmalonyl-CoA-decarboxylase with a specific activity of about 5 U/mg of protein was isolated by adsorption and elution from monomeric avidin-Sepharose. To analyze the function of the ␦ and subunits, the corresponding genes were deleted from plasmid pT7mmd. E. coli cells transformed with pJH⌬2, which lacks mmdE and the 3-terminal part of mmdD, showed no methylmalonyl-CoA-decarboxylase activity. In addition, a core complex consisting of the remaining subunits (␣, ␤, and ␥) could not be isolated from this mutant. In contrast, catalytically active methylmalonyl-CoA-decarboxylase was expressed in E. coli from plasmid pJH⌬1, which contained a deletion of the mmdE gene only. The mutant enzyme could be isolated, reconstituted into proteoliposomes, and shown to function in the transport of Na ؉ ions coupled to methylmalonyl-CoA decarboxylation. The small subunit therefore has no catalytic function within the methylmalonyl-CoA-decarboxylase complex but appears to increase the stability of this complex.

Energy conservation in the decarboxylation of dicarboxylic acids by fermenting bacteria

“…The MmdE protein is part of the methylmalonyl-CoA decarboxylase complex of V. parvula. It has no catalytic function but increases the stability of the complex, as has been shown by comparing the stability of the five-subunit and the four-subunit complex after production from appropriate plasmids in E. coli (Huder and Dimroth 1995). These data are consistent with the fact that the four-subunit methylmalonyl-CoA decarboxylase of P. modestum is functional but exhibits poor complex stability.…”

The KdpF Subunit Is Part of the K+-translocating Kdp Complex of Escherichia coli and Is Responsible for Stabilization of the Complex in Vitro

“…As in the case of KdpF, this subunit is not essential in vivo but stabilizes the enzyme complex in vitro, especially the interaction of the membrane-bound part with the soluble ␣ subunit. The affinity for substrate and inhibitors of a complex missing subunit ⑀ is not impaired (53). Interestingly, the methylmalonyl-CoA-decarboxylase from Propionigenium modestum lacks the ⑀ subunit, leading to a relatively unstable enzyme complex during purification (54).…”

The Rumen Microbial Ecosystem

“…Like M. elsdenii, V. parvula possesses phospholipids containing phosphatidylserine, phosphatidylethanolamine and plasmalogens (Johnston and Goldfine, 1982). In V. parvula, the terminal step in succinate decarboxylation, the conversion of S-methylmalonyl-CoA to CO2 and propionyl-CoA, is catalysed by methylmalonyl-CoA decarboxylase, which acts as a sodium pump, exporting Na+ and conserving the decarboxylation energy as a sodium gradient (Huder and Dimroth, 1995). When succinate is present as co-substrate, growth yields of V. parvula increased, probably because the sodium gradient saves metabolic energy which would otherwise be expended in processes such as substrate uptake (Denger and Schink, 1992).…”