Primary structure of the 5 S subunit of transcarboxylase as deduced from the genomic DNA sequence

FEBS Letters

Phillips, Nelson F.B.¹  Shenoy, Bhami C.¹  Magner, William J.¹  Thornton, Charles G.¹  Wood, Harland G.¹  Hejlik, Daniel P.¹  Park, Vicki M.¹  Haase, F.Carl¹  Samols, David¹  Kumar, Ganesh K.¹ 

[1] Department of Biochemistry, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4935, USA

关键词: Transcarboxylase;    Biotin;    Pyruvate;    Oxalacetate;    Sequence;    5 S subunit;    TC transcarboxylase;    WT wild type;    MMCoA;    methylmalonyl-CoA;    Bct;    biocytin;   

DOI  :  10.1016/0014-5793(93)80271-U

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown