Rescue of protein splicing activity from a Magnetospirillum magnetotacticum intein-like element.
The self-catalytic protein splicing mechanism is mediated by the intein plus the first amino acid following the intein C-terminus (termed the +1 residue). Although polymorphisms of conserved residues elsewhere in inteins have been widely reported, no splicing-competent intein has been observed without a Ser, Thr or Cys in this functionally essential +1 position. This residue is the nucleophile in two steps of the protein splicing pathway: ligation of the extein fragments during transesterification and formation of a peptide bond between the exteins by an acyl rearrangement. An intein-like element in a hypothetical protein (gene Magn8951) from Magnetospirillum magnetotacticum has all intein signature sequences except the +1 residue, where it has a Tyr. Although the Tyr side-chain hydroxyl can potentially mediate the transesterification reaction, an acyl shift has never been observed with this residue. When the activities of this bacterial intein-like element were studied, protein splicing was not observed and N-terminal cleavage predominated. Mutation of Tyr+1 to Phe or Ala indicated that the Tyr side-chain hydroxyl was not necessary for N-terminal cleavage. Protein splicing activity could be rescued by "reversion" of Tyr+1 to Cys.
Southworth
M W
MW
New England BioLabs, 32 Tozer Road, Beverly, MA 01915, U.S.A.
Yin
J
J
Perler
F B
FB
D000409:Alanine / Q000737:chemistry; D017398:Alternative Splicing; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry; D042201:Magnetospirillum / Q000378:metabolism*; D008957:Models, Genetic; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D011110:Polymorphism, Genetic; D019154:Protein Splicing; D017434:Protein Structure, Tertiary; D012326:RNA Splicing; D012694:Serine / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D014443:Tyrosine; D012694:Serine; D010649:Phenylalanine; D003545:Cysteine; D000409:Alanine