Expanding the definition of class 3 inteins and their proposed phage origin

Inteins are the protein equivalent of introns. Their protein splicing activity is essential for the host protein's maturation and function. Inteins are grouped into three classes based on sequence signature and splicing mechanism. The sequence signature of the recently characterized class 3 inteins is a noncontiguous Trp-Cys-Thr (WCT) motif and the absence of the standard class 1 Cys¹ or Ser¹ N-terminal nucleophile. The intein N-terminal Cys¹ or Ser¹ residue is essential for splicing in class 1 inteins. The mycobacteriophage Catera Gp206, Nocardioides sp. strain JS614 TOPRIM, and Thermobifida fusca YX Tfu2914 inteins have a mixture of class 1 and class 3 motifs. They carry the class 3 Trp-Cys-Thr motif and have the standard class 1 N-terminal Ser¹ or Cys¹. This study determined which class the mycobacteriophage Catera Gp206 and Nocardioides sp. JS614 TOPRIM inteins belong to based on catalytic mechanism. The mycobacteriophage Catera Gp206 intein (starting with Ser¹) is a class 3 intein, and its Ser¹ residue is not required for splicing. Based on phylogenetic analysis, we propose that class 3 inteins arose from a single mutated intein that was spread by phage into predominantly helicase genes in various phages and their hosts.