STRUCTURAL CHARACTERIZATION OF THE ENTIRE 1.3S SUBUNIT OF TRANSCARBOXYLASE FROM PROPIONIBACTERIUM-SHERMANII

Transcarboxylase (TC) from Propionibacterium shermanii, a biotin-depen dent enzyme, catalyzes the transfer of a carboxyl group from methylmal onyl-CoA to pyruvate in two partial reactions. Within the multisubunit enzyme complex, the 1.3S subunit functions as the carboxyl group carr ier. The 1.3S is a 123-amino acid polypeptide (12.6 kDa), to which bio tin is covalently attached at Lys 89. We have expressed 1.3S in Escher ichia coli with uniform N-15 labeling. The backbone structure and dyna mics of the protein have been characterized in aqueous solution by thr ee-dimensional heteronuclear nuclear magnetic resonance (NMR) spectros copy. The secondary structure elements in the protein were identified based on NOE information, secondary chemical shifts, homonuclear (3)J( HNH alpha), coupling constants, and amide proton exchange data. The pr otein contains a predominantly disordered N-terminal half, while the C -terminal half is folded into a compact domain comprising eight beta-s trands connected by short loops and turns. The topology of the C-termi nal domain is consistent with the fold found in both carboxyl carrier and lipoyl domains, to which this domain has approximately 26-30% sequ ence similarity.