Format

Send to

Choose Destination
Biochemistry. 2012 Mar 27;51(12):2496-505. doi: 10.1021/bi201840k. Epub 2012 Mar 13.

The Thermococcus kodakaraensis Tko CDC21-1 intein activates its N-terminal splice junction in the absence of a conserved histidine by a compensatory mechanism.

Author information

1
New England BioLabs, Ipswich, Massachusetts 01938, United States.

Abstract

Inteins and other self-catalytic enzymes, such as glycosylasparaginases and hedgehog precursors, initiate autocleavage by converting a peptide bond to a (thio)ester bond when Ser, Thr, or Cys undergoes an N-[S/O] acyl migration assisted by residues within the precursor. Previous studies have shown that a His at position 10 in intein Block B is essential for this initial acyl migration and N-terminal splice junction cleavage. This His is present in all inteins identified to date except the Thermococcus kodakaraensis Tko CDC21-1 intein orthologs and the inactive Arthrobacter species FB24 Arth_1007 intein. This study demonstrates that the Tko CDC21-1 intein is fully active and has replaced the lost catalytic function normally provided by the Block B His using a compensatory mechanism involving a conserved ortholog-specific basic residue (Lys(58)) present outside the standard intein conserved motifs. We propose that Lys(58) catalyzes the initial N-S acyl migration by stabilizing the thiazolidine-tetrahedral intermediate, allowing it to be resolved by water-mediated hydrolysis rather than by protonating the leaving group as His is theorized to do in many other inteins. Autoprocessing enzymes may have more flexibility in evolving catalytic variations because high reaction rates are not required when performing single-turnover reactions on "substrates" that are covalently attached to the enzyme. Consequently, inteins have more flexibility to sample catalytic mechanisms, providing insight into various strategies that enzymes use to accomplish catalysis.

PMID:
22380677
DOI:
10.1021/bi201840k
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for American Chemical Society
Loading ...
Support Center