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.
A naturally occurring split intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) has been shown to mediate efficient in vivo and in vitro transsplicing in a foreign protein context. A cis-splicing Ssp DnaE intein construct displayed splicing activity similar to the trans-splicing form, which suggests that the Nand C-terminal intein fragments have a high affinity interaction. An in vitro trans-splicing system was developed that used a bacterially expressed N-terminal fragment of the Ssp DnaE intein and either a bacterially expressed or chemically synthesized intein C-terminal fragment. Unlike artificially split inteins, the Ssp DnaE intein fragments could be reconstituted in vitro under native conditions to mediate splicing as well as peptide bond cleavage. This property allowed the development of an on-column trans-splicing system that permitted the facile separation of reactants and products. Furthermore, the trans-splicing activity of the Ssp DnaE intein was successfully applied to the cyclization of proteins in vivo. Also, the isolation of the unspliced precursor on chitin resin allowed the cyclization reaction to proceed in vitro. The Ssp DnaE intein thus represents a potentially important protein for in vivo and in vitro protein manipulation.Protein splicing elements, termed inteins (1), catalyze their own excision from a primary translation product with the concomitant ligation of the flanking protein sequences (reviewed in Refs. 2-4). Inteins catalyze three highly coordinated reactions at the N-and C-terminal splice junctions (5, 6): 1) an acyl rearrangement at the N-terminal cysteine or serine; 2) a transesterification reaction between the two termini to form a branched ester or thioester intermediate; and 3) peptide bond cleavage coupled to cyclization of the intein C-terminal asparagine to free the intein. Inteins have been engineered to be versatile tools in protein purification (7-13), protein ligation (9, 10, 12, 14 -18), and in the formation of cyclic proteins and peptides (11,19,20). However, the ligation and cyclization approaches were limited by the need to generate an N-terminal cysteine and/or C-terminal thioester intermediate in vitro.In addition to inteins engineered to trans-splice (21-24), a naturally occurring split intein was recently identified in the dnaE gene encoding the catalytic subunit of DNA polymerase III of Synechocystis sp. PCC6803 (25). The N-terminal half of DnaE, followed by a 123-amino acid intein sequence, and the C-terminal half, preceded by a 36-amino acid intein sequence, are encoded by two open reading frames located more than 745 kilobases apart in the genome. When co-expressed in Escherichia coli, the two DnaE-intein fragments exhibited protein trans-splicing (25). In this report we have further investigated the cis-and trans-splicing activities of the Ssp DnaE intein in a foreign protein context. Furthermore, novel methods were developed that allow the on-column ligation of protein fragments as well as the in vivo and in vitro cyclization of p...
The identity and amounts of intracellular solutes in the deep-sea bacterium Photobacterium profundum strain SS9 were studied using nuclear magnetic resonance techniques. P. profundum strain SS9, a moderate piezophile which grows optimally at 20-30 MPa primarily accumulated glutamate and betaine, with lesser amounts of alanine, beta-hydroxybutyrate (beta-HB) and oligomers composed of the beta-HB units when grown at 0.1 MPa to early stationary phase. When grown at the optimal pressure, the cells preferentially increased intracellular concentrations of beta-HB and beta-HB oligomers, while the amino acid pools remained relatively constant. Since the organic solutes increased with increasing external NaCl in the medium, they are functioning as osmolytes. The beta-HB molecules represent a novel class of osmolytes, termed 'piezolytes,' whose cellular levels responded to hydrostatic pressure as well as osmotic pressure. Factors such as cell growth stage and temperature were also examined for their effect on the solute distribution in these cells.
Use of the naturally split, self-splicing Synechocystis sp. PCC6803 DnaE intein permits separate purification of the N- and C-terminal intein domains. Otherwise spontaneous intein-mediated reactions can therefore be controlled in vitro, allowing detailed study of intein kinetics. Incubation of the Ssp DnaE intein with ZnCl(2) inhibited trans splicing, hydrolysis-mediated N-terminal trans cleavage, and C-terminal trans cleavage reactions. Maximum inhibition of the splicing reaction was achieved at equal molar concentrations of ZnCl(2) and intein domains, suggesting a 1:1 metal ion:intein binding stoichiometry. Mutation of the (+)1 cysteine residue to valine (C(+)1V) alleviated the inhibitory effects of ZnCl(2). Valine substitution in the absence of ZnCl(2) blocked trans splicing and decreased C-terminal cleavage kinetics in a manner similar to that of the native (+)1 cysteine in the presence of ZnCl(2). These data are consistent with Zn(2+)-mediated inhibition of the Ssp DnaE intein via chelation of the (+)1 cysteine residue. N-Terminal trans cleavage can occur via both spontaneous hydrolysis and nucleophilic (e.g., DTT) attack. Comparative examination of N-terminal cleavage rates using amino acid substitution (C(+)1V) and Zn(2+)-mediated inhibition permitted the maximum contribution of hydrolysis to overall N-terminal cleavage kinetics to be determined. Stable intermediates consisting of the associated intein domains were detected by PAGE and provided evidence of a rapid C-terminal cleavage step. Acute control of the C-terminal reaction was achieved by the rapid reversal of Zn(2+)-mediated inhibition by EDTA. By inhibiting both the splicing pathway and spontaneous hydrolysis with Zn(2+), reactants can be diverted from the trans splicing to the trans cleavage pathway where DTT and EDTA can regulate N- and C-terminal cleavage, respectively.
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