Abstract
An Arthrobacter species FB24 gene (locus tag Arth_1007) was previously annotated as a putative intein-containing DnaB helicase of phage origin (Arsp-FB24 DnaB intein). However, it is not a helicase gene because the sequence similarity is limited to inteins. In fact, the flanking exteins total only 66 amino acids. Therefore, the intein should be referred to as the Arsp-FB24 Arth_1007 intein. The Arsp-FB24 Arth_1007 intein failed to splice in its native precursor and in a model precursor. We previously noted that the Arsp-FB24 Arth_1007 intein is the only putative Class 3 intein that is missing the catalytically essential Cys at position 4 of intein Motif F, which is one of the three defining signature residues of this class. Additionally, a catalytically essential His in position 10 of intein Motif B is also absent; this His is the most conserved residue amongst all inteins. Splicing activity was not rescued when these two catalytically important positions were ‘reverted’ back to their consensus residues. This study restores the unity of the Class 3 intein signature sequence in active inteins by demonstrating that the Arsp-FB24 Arth_1007 intein is an inactive pseudogene.
Highlights
Inteins are protein splicing elements that remove themselves from host proteins during post-translational processing
Some inteins are chimeric proteins with a centrally located homing endonuclease domain containing four endonuclease motifs (C, D, E, and H, Figure 1) [2,3,4]. The His at position 10 of intein Motif B (B:10) is the most conserved intein amino acid and is essential for splicing in all inteins previously tested [1,5,6,8,9,10,11]; it is an Asn in the Arthrobacter species FB24 (Arsp-FB24) Arth_1007 (DnaB) intein
The Arsp-FB24 Arth_1007 intein with 5 native extein residues on both sides was cloned by PCR into a model precursor termed MIP, with the intein flanked by the E. coli Maltose Binding Protein (M) and the DSal fragment of D. immitis paramyosin (P) as previously described [1,10,11,13]
Summary
Inteins are protein splicing elements that remove themselves from host proteins (exteins) during post-translational processing. Most inteins are Class 1 inteins that splice themselves out of precursor proteins by a four-step mechanism [5,7,12] consisting of an initial acyl shift of the intein N-terminal Ser, Thr or Cys to form a linear (thio)ester intermediate, followed by a transesterification reaction that results in a branched intermediate (Figure 2). This branched intermediate is resolved by cyclization of the intein Cterminal Asn, which separates the intein from the ligated exteins. This suggests that either it is an inactive intein or it is not a Class 3 intein
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