Characterization of a naturally occurring trans-splicing intein from Synechocystis sp PCC6803

A naturally occurring trans-splicing intein from the dnaE gene of Synechocy stis sp. PCC6803 (Ssp DnaE intein) was used to characterize the intein-cata lyzed splicing reaction. Trans-splicing/cleavage reactions were initiated b y combining the N-terminal splicing domain of the Ssp DnaE intein containin g five native N-extein residues and maltose binding protein as the N-extein with the C-terminal Ssp DnaE intein splicing domain (E-C) with or without thioredoxin fused in-frame to its carboxy terminus. Observed rate constants (k(obs)) for dithiothreitol-induced N-terminal cleavage, C-terminal cleava ge, and trans-splicing were (1.0 +/- 0.5) x 10(-3), (1.9 +/- 0.9) x 10(-4), and (6.6 +/- 1.3) x 10(-5) s(-1), respectively. Preincubation of the intei n fragments showed no change in k(obs), indicating association of the two s plicing domains is rapid relative to the subsequent steps. Interestingly, w hen E-C concentrations were substoichiometric with respect to the N-termina l splicing domain, the levels of N-terminal cleavage were equivalent to the amount of E-C, even over a 24 h period. Activation energies for N-terminal cleavage and trans-splicing were determined by Arrhenius plots to be 12.5 and 8.9 kcal/mol, respectively. Trans-splicing occurred maximally at pH 7.0 , while a slight increase in the extent of N-terminal cleavage was observed at higher pH values. This work describes an in-depth kinetic analysis of t he splicing and cleavage activity of an intein, and provides insight for th e use of the split intein as an affinity domain.