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J. Bacteriol., 07 1995, 3623-3630, Vol 177, No. 13
Copyright © 1995, American Society for Microbiology

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

JB Huder and P Dimroth
Mikrobiologisches Institut, Eidgenossischen Technischen Hochschule, Zurich, Switzerland.

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-CoA-decarboxylase, 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 gamma subunit was only partially biotinylated and the alpha 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 alpha 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 delta and epsilon subunits, the corresponding genes were deleted from plasmid pT7mmd.(ABSTRACT TRUNCATED AT 250 WORDS)

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