Primary structure of the 5 S subunit of transcarboxylase as deduced from the genomic DNA sequence.
Transcarboxylase from Propionibacterium shermanii is a complex biotin-containing enzyme composed of 30 polypeptides of three different types. It is composed of six dimeric outer subunits associated with a central cylindrical hexameric subunit through 12 biotinyl subunits; three outer subunits on each face of the central hexamer. Each outer dimer is termed a 5 S subunit which associates with two biotinyl subunits. The enzyme catalyzes a two-step reaction in which methylmalonyl-CoA and pyruvate form propionyl-CoA and oxalacetate, the 5 S subunit specifically catalyzing one of these reactions. We report here the cloning, sequencing and expression of the monomer of the 5 S subunit. The gene was identified by matching amino acid sequences derived from isolated authentic 5 S peptides with the deduced sequence of an open reading frame present on a cloned P. shermanii genomic fragment known to contain the gene encoding the 1.3 S biotinyl subunit. The cloned 5 S gene encodes a protein of 519 amino acids, M(r) 57,793. The deduced sequence shows regions of extensive homology with that of pyruvate carboxylase and oxalacetate decarboxylase, two enzymes which catalyze the same or reverse reaction. A fragment was subcloned into pUC19 in an orientation such that the 5 S 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 which co-migrated with authentic 5 S and were fully active in catalyzing the 5 S partial reaction. We conclude that we have cloned, sequenced and expressed the monomer of the 5 S subunit and that the expressed product is catalytically active.
Thornton
C G
CG
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4935.
Kumar
G K
GK
Shenoy
B C
BC
Haase
F C
FC
Phillips
N F
NF
Park
V M
VM
Magner
W J
WJ
Hejlik
D P
DP
Wood
H G
HG
Samols
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019878:Carboxyl and Carbamoyl Transferases; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli; D008969:Molecular Sequence Data; D011424:Propionibacterium / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D014166:Transferases / Q000235:genetics*
D004269:DNA, Bacterial; D014166:Transferases; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase