Characterization of a naturally occurring trans-splicing intein from Synechocystis sp. PCC6803.
A naturally occurring trans-splicing intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) was used to characterize the intein-catalyzed splicing reaction. Trans-splicing/cleavage reactions were initiated by combining the N-terminal splicing domain of the Ssp DnaE intein containing 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 cleavage, 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 intein fragments showed no change in k(obs), indicating association of the two splicing domains is rapid relative to the subsequent steps. Interestingly, when E(C) concentrations were substoichiometric with respect to the N-terminal 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 the splicing and cleavage activity of an intein, and provides insight for the use of the split intein as an affinity domain.
Martin
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New England Biolabs, Inc., Beverly, Massachusetts 01915-5599, USA.
Xu
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Evans
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D000595:Amino Acid Sequence; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004229:Dithiothreitol / Q000737:chemistry; D004951:Esterification; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D019811:Hydroxylamine / Q000737:chemistry; D007700:Kinetics; D015080:Mesna / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D019154:Protein Splicing; D012680:Sensitivity and Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D020040:Trans-Splicing
D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D019811:Hydroxylamine; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III; D015080:Mesna; D004229:Dithiothreitol