Purification and characterization of the recombinant 5 S subunit of transcarboxylase from Escherichia coli.
Transcarboxylase from Propionibacterium shermanii is a biotin-containing enzyme which catalyzes the reversible transfer of a carboxyl group from methylmalonyl-CoA to pyruvate. It is composed of a central, hexameric 12 S subunit, 6 outer dimeric 5 S subunits which are held in a complex by 12 1.3 S biotinyl subunits. The transcarboxylase reaction requires two partial reactions, one of which is specific to 5 S. The cloning and expression of each of these subunits in Escherichia coli have been reported. We have designed a method for the purification of the 5 S subunit from an E. coli expression system. Protein purified to homogeneity by this method was shown to be active in the 5 S partial reaction, but unable to catalyze the overall transcarboxylase reaction. This protein was characterized as to its ability to form stable dimers, associate with the 1.3 S subunit in stable complexes referred to as 6 S, and assemble whole TC. The latter activity was shown to be lacking. The purified protein has a native molecular weight of 120 kDa and a subunit molecular weight of 60 kDa, consistent with the 5 S dimer. Plasma emission analysis of the metal content of the recombinant protein demonstrated the presence of both Co and Zn, comparable to the authentic protein. Fluorescence analysis verified the ability of the purified protein to bind substrates and 1.3 S subunits appropriately. Sequencing of the amino terminus and determination of the amino acid composition of the recombinant protein relative to that of the authentic subunit further verified the identity of the purified protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Xie
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Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106-4935.
Shenoy
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BC
Magner
W J
WJ
Hejlik
D P
DP
Samols
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D
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D019878:Carboxyl and Carbamoyl Transferases; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014166:Transferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D014166:Transferases; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase