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Literature summary for 3.4.21.53 extracted from

  • OBrien, K.M.; Schufreider, A.K.; McGill, M.A.; OBrien, K.M.; Reitter, J.N.; Mills, K.V.
    Mechanism of protein splicing of the Pyrococcus abyssi lon protease intein (2010), Biochem. Biophys. Res. Commun., 403, 457-461.
    View publication on PubMed
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Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli of intein fusion protein that consists of an N-terminal poly-His tag followed by Escherichia coli MBP, the 23 C-terminal residues of the N-extein, the 333 residues of the lon protease intein, the 27 N-terminal residues of the C-extein, and a Cterminal GST Pyrococcus abyssi

Protein Variants

Protein Variants Comment Organism
D93A mutations result in efficient splicing Pyrococcus abyssi
H94A mutation results in the accumulation of precursor Pyrococcus abyssi
K332A mutation results mostly in splicing, with some accumulation of branched-ester intermediate Pyrococcus abyssi
K332H mutation results mostly in splicing, with some accumulation of branched-ester intermediate Pyrococcus abyssi
K332R mutation results in splicing as efficient as wild-type Pyrococcus abyssi
additional information the lon protease of Pyrococcus abyssi is interupted by an intein. The intein splices essentially to completion when over-expressed in Escherichia coli. Blocking the first step of splicing with a Cys1 to Ala mutation or step two of splicing with a Ser+1 to Ala mutation leads to the accumulation of precursor. Substitution of Ser+1 with Thr results in precursor, whereas substitution to Cys results in efficient splicing. The influence of the flanking extein residues on splicing efficiency is as follows. Mutation of Gly+2, Gly+3, Gly+5 or Gly+2/Gly+3 to Ala results mostly in splicing, but mutation of Gly+2 also results in the accumulation of branched-ester intermediate. The identity of the C-terminal residue of the N-extein seems less important, as mutation of Gln1 to Asn, Ala, Glu or Gly results in efficient splicing Pyrococcus abyssi
N333A prevention of step three of the intein splicing, mutation results in the accumulation of precursor and branched-ester intermediate Pyrococcus abyssi
P92A mutations results in efficient splicing Pyrococcus abyssi
T91A mutation results in the accumulation of precursor Pyrococcus abyssi

Organism

Organism UniProt Comment Textmining
Pyrococcus abyssi
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Subunits

Subunits Comment Organism
More the lon protease of Pyrococcus abyssi is interupted by an intein. The intein splices essentially to completion when over-expressed in Escherichia coli. Blocking the first step of splicing with a Cys1 to Ala mutation or step two of splicing with a Ser+1 to Ala mutation leads to the accumulation of precursor. Substitution of Ser+1 with Thr results in precursor, whereas substitution to Cys results in efficient splicing. Prevention of step three of splicing by mutation of the intein C-terminal Asn333 to Ala results in the accumulation of precursor and branched-ester intermediate Pyrococcus abyssi