Primary structure of the monomer of the 12S subunit of transcarboxylase as deduced from DNA and characterization of the product expressed in Escherichia coli.
Transcarboxylase from Propionibacterium shermanii is a complex biotin-containing enzyme composed of 30 polypeptides of three different types: a hexameric central 12S subunit to which 6 outer 5S subunits are attached through 12 1.3S biotinyl subunits. The enzyme catalyzes a two-step reaction in which methylmalonyl coenzyme A and pyruvate serve as substrates to form propionyl coenzyme A (propionyl-CoA) and oxalacetate, the 12S subunit specifically catalyzing one of the two reactions. We report here the cloning, sequencing, and expression of the 12S subunit. The gene was identified by matching amino acid sequences derived from isolated authentic 12S peptides with the deduced sequence of an open reading frame present in a cloned P. shermanii genomic fragment known to contain the gene encoding the 1.3S biotinyl subunit. The cloned 12S gene encodes a protein of 604 amino acids and of M(r) 65,545. The deduced sequence shows regions of extensive homology with the beta subunit of mammalian propionyl-CoA carboxylase as well as regions of homology with acetyl-CoA carboxylase from several species. Two genomic fragments were subcloned into pUC19 in an orientation such that the 12S open reading frame could be expressed from the lac promoter of the vector. Crude extracts prepared from these cells contained an immunoreactive band on Western blots (immunoblots) which comigrated with authentic 12S. The Escherichia coli-expressed 12S was purified to apparent homogeneity by a three-step procedure and compared with authentic 12S from P. shermanii. Their quaternary structures were identical by electron microscopy, and the E. coli 12S preparation was fully active in the reactions catalyzed by this subunit. We conclude that we have cloned, sequenced, and expressed the 12S subunit which exists in a hexameric active form in E.coli.
Thornton
C G
CG
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106-4935.
Kumar
G K
GK
Haase
F C
FC
Phillips
N F
NF
Woo
S B
SB
Park
V M
VM
Magner
W J
WJ
Shenoy
B C
BC
Wood
H G
HG
Samols
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019878:Carboxyl and Carbamoyl Transferases; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D011424:Propionibacterium / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014166:Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004269:DNA, Bacterial; D014166:Transferases; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase