CRYSTAL-STRUCTURE OF GYRA INTEIN FROM MYCOBACTERIUM-XENOPI REVEALS STRUCTURAL BASIS OF PROTEIN SPLICING

Several genes from prokaryotes and lower eukaryotes have been found to contain an in-frame open reading frame, which encodes for an internal protein (intein). Post-translationally, the internal polypeptide auto -splices and ligates the external sequences to yield a functional exte rnal protein (extein) and an intein. Most, but not all inteins, contai n, apart from a splicing domain, a separate endonucleolytic domain tha t enables them to maintain their presence by a homing mechanism, We re port here the crystal structure of an intein found in the gyrase A sub unit from Mycobacterium xenopi at 2.2 Angstrom resolution, The structu re contains an unusual P-fold with the catalytic splice junctions at t he ends of two adjacent antiparallel P-strands. The arrangement of the active site residues Ser 1, Thr 72, His 75, His 197, and Asn 198 is c onsistent with a four-step mechanism for the cleavage-ligation reactio n. Using site-directed mutagenesis, the N-terminal cysteine, proposed as the nucleophile in the first step of the splicing reaction, was cha nged to a Ser 1 and Ala 0, thus capturing the intein in a pre-spliced state.