Abstract
Inteins are the protein equivalent of introns. They are seamlessly removed during post-translational maturation of their host protein (extein). Inteins from extremophiles played a key role in understanding intein-mediated protein splicing. There are currently three classes of inteins defined by catalytic mechanism and sequence signatures. This study demonstrates splicing of three class 3 mini-inteins: Burkholderia vietnamiensis G4 Bvi IcmO intein, Mycobacterium smegmatis MC2 155 Msm DnaB-1 intein and Mycobacterium leprae strain TN Mle DnaB intein. B. vietnamiensis has a broad ecological range and remediates trichloroethene. M. smegmatis is a biofilm forming soil bacteria. Although other intein classes have only a single branched intermediate at the C-terminal splice junction, the class 3 intein reaction pathway includes two branched intermediates. The class 3 specific branched intermediate is formed by an internal cysteine, while the C-terminal branch intermediate is at a serine or threonine in all class 3 inteins except the Bvi IcmO intein, where it is a cysteine. This latter cysteine was unable to compensate for mutation of the class 3-specific internal catalytic cysteine despite the Bvi IcmO intein having an N-terminal splice junction naturally tuned for a cysteine nucleophile, demonstrating the mandatory order of branch intermediates in class 3 inteins.
Highlights
Inteins are protein splicing elements that are removed from precursor proteins by a self-catalytic mechanism
DNAs encoding the Msm DnaB-1 (139 aa) and Mle DnaB (145 aa) mini-inteins along with 5 DnaB extein residues flanking the intein on each side (Perler 2002) were synthesized and cloned in the MIP model precursor system (Xu et al 1994) between the E. coli maltose binding protein (MBP or M) and the D. immitis paramyosin Δ Sal fragment (P) generating precursors MSP and MLP, respectively
MVP spliced poorly with less than half of the MVP precursor converted to spliced product at all temperatures tested (Fig. 3 and data not shown). These results demonstrate that all three mini-inteins are active, the degree of splicing in these model precursors varied with the intein
Summary
Inteins are protein splicing elements that are removed from precursor proteins by a self-catalytic mechanism. Extremophiles (2017) 21:41–49 known mechanisms of intein-mediated protein splicing (Brace et al 2010; Eryilmaz et al 2014; Mills et al 2014; Southworth et al 2000; Tori et al 2010; Volkmann and Mootz 2013; Xu et al 1994; Xu and Perler 1996). The intein and extein are translated as a single, fused precursor protein. The majority of inteins are bifunctional enzymes that have a homing endonuclease domain as well as a protein splicing domain. The homing endonuclease is responsible for lateral transmission of intein genes, making them parasitic mobile genetic elements (Barzel et al 2011; Novikova et al 2014). Mini-inteins do not have an endonuclease domain, but retain the core protein splicing domain. Modern day mini-inteins are thought to be the descendents of inteins that lost their homing endonuclease domain (Barzel et al 2011; Novikova et al 2014)
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