Inteins in Microbial Genomes: Distribution, Mechanism, and Function

Inteins are self-splicing protein elements (134 to 608 amino acids). Over 125 inteins have been cataloged in InBase, the on-line intein database (http://www.neb.com/neb/inteins.html), which includes the Intein Registry[1]. Inteins naturally present in pathogenic microbes represent novel, yet unexploited drug targets. Understanding the chemistry of the splicing reaction has allowed the manipulation of inteins, which are now used in many protein engineering applications[2]

The Arthrobacter Species FB24 Arth_1007 (DnaB) Intein Is a Pseudogene

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

Protein Splicing of the Deinococcus radiodurans Strain R1 Snf2 Intein

Adjacent intein fragments fused to a Snf2/Rad54 helicase-related protein and Snf2/Rad54 helicase were reported for Deinococcus radiodurans R1, leading to the speculation that a frameshift was required for splicing or that trans splicing occurred. However, a type strain (ATCC 13939, RF18410) yielded a single protein that splices by the Ala1 protein splicing pathway, with splicing dependent on adjacent residues

An alternative protein splicing mechanism for inteins lacking an N-terminal nucleophile

Variations in the intein-mediated protein splicing mechanism are becoming more apparent as polymorphisms in conserved catalytic residues are identified. The conserved Ser or Cys at the intein N-terminus and the conserved intein penultimate His are absent in the KlbA family of inteins. These inteins were predicted to be inactive, since an N-terminal Ala cannot perform the initial reaction of the standard protein splicing pathway to yield the requisite N-terminal splice junction (thio)ester. Despite the presence of an N-terminal Ala and a penultimate Ser, the KlbA inteins splice efficiently using an alternative protein splicing mechanism. In this non-canonical pathway, the C-extein nucleophile attacks a peptide bond at the N-terminal splice junction rather than a (thio)ester bond, alleviating the need to form the initial (thio)ester at the N-terminal splice junction. The remainder of the two pathways is the same: branch resolution by Asn cyclization is followed by an acyl rearrangement to form a native peptide bond between the ligated exteins

Sequence alignment of the Arsp-FB24 Arth_1007 intein (Arsp) vs. the MP-Catera Gp206 intein (Catera).

<p>Conserved splicing motifs (A, B, F, and G) and homing endonuclease motifs (C, D, E and H), as described in the InBase intein database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026361#pone.0026361-Perler1" target="_blank">[2]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026361#pone.0026361-Pietrokovski1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026361#pone.0026361-Pietrokovski2" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026361#pone.0026361-Perler2" target="_blank">[6]</a>, are indicated above the Arsp-FB24 Arth_1007 intein sequence. Positions within each motif are numbered from amino to carboxy terminal and are referred to using the motif letter and the position number separated by a colon. Arsp-FB24 Arth_1007 intein residues Asn⁶⁵ in Motif B position 10 (B∶10) and Gly³¹¹ in Motif F position 4 (F∶4) are underlined. Residues present in both inteins are listed and similar substitutions are marked with a plus sign.</p

The native Arsp-FB24 Arth_1007 precursor in inactive.

<p>The native Arsp-FB24 Arth_1007 precursor (NIC) was expressed in E.coli with the addition of an N-terminal His tag. Because the C-extein is only 14 residues, a precursor truncated at the intein C-terminus (NI) was also expressed. The left panel is a SDS-PAGE of soluble lysates after in vivo expression at 37°C stained with Simply Blue Safe Stain and the right panel is a Western blot of the same samples run in the same gel. Proteins containing the N-terminal His tag were detected with the anti-His tag antibody. The control lane (CTR) contains soluble lysates from E.coli with just an empty plasmid. The sizes of the molecular weight standards (M) are listed in kDa (New England Biolabs 10 to 250 kDa ladder).</p