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Primary structure of the 5 S subunit of transcarboxylase as deduced from the genomic DNA sequence
Author(s) -
Thornton Charles G.,
Kumar Ganesh K.,
Shenoy Bhami C.,
Haase F.Carl,
Phillips Nelson F.B.,
Park Vicki M.,
Magner William J.,
Hejlik Daniel P.,
Wood Harland G.,
Samols David
Publication year - 1993
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(93)80271-u
Subject(s) - protein subunit , biology , microbiology and biotechnology , biochemistry , open reading frame , protein primary structure , peptide sequence , genomic dna , gene
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.