Attacking from the rear. Unlike traditional intein splicing mechanisms (right), the KlbA inteins make a direct nucleophilic attack on the N-terminal splice junction near the first extein (1) with a cysteine side chain next to the second extein (2). This eliminates the transesterification step of the traditional mechanism. (Adapted from Southworth, M. W., et al. EMBO J. 2000, 19, 5019–5026.)

This autocatalytic process of protein maturation is widespread. More than 100 inteins have been identified in protein precursors isolated from various organisms, including viruses and bacteria.

The standard four-step mechanism of protein splicing hinges on a nucleophilic serine or cysteine located at the intein’s N-terminus and is facilitated by its penultimate histidine. These residues are highly conserved and, according to the canonical view, inteins that lack these residues should be inactive. Recently, researchers from New England BioLabs in Beverly, MA, described an alternative mechanism for protein splicing, which takes place in the KlbA family of archaeal inteins and features an N-terminal alanine and penultimate serine (EMBO J. 2000, 19, 5019–5026).

In this unusual but efficient pathway, the N-terminal cysteine of the second extein acts as a nucleophile and directly attacks the peptide bond at the N-terminal splice junction. This removes the need for an intermediate (thio)ester between an intein’s nucleophile and the splice junction. As a result, the direct attack by the second extein saves one step in the overall process, the transesterification.

Although it is still unclear why other inteins cannot perform similar reactions, the researchers have begun experiments to address this question.