title: Sequential formation of two branched intermediates during protein splicing of class three inteins.
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.
authors.affiliation: New England Biolabs, Inc., Ipswich, MA, 01938, USA.|Takara Bio USA, Inc., 1290 Terra Bella Ave., Mountain View, CA, 94043, USA.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Tori
authors.affiliation: New England Biolabs, Inc., Ipswich, MA, 01938, USA. PerlsOfWisdom@icloud.com.|Perls of Wisdom Biotech Consulting, Brookline, MA, 02446, USA. PerlsOfWisdom@icloud.com.|Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 06520, USA. PerlsOfWisdom@icloud.com.
authors.forename: Francine
authors.initials: F
authors.lastname: Perler
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D019117:Burkholderia / Q000235:genetics / Q000378:metabolism*; D047668:Inteins; D009166:Mycobacterium leprae / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
chemical_list: D001426:Bacterial Proteins
keywords: Branched intermediate; Cysteine; Enzyme mechanism; Intein; Protein splicing