13 research outputs found
Inteins in Microbial Genomes: Distribution, Mechanism, and Function
Inteins are self-splicing protein elements (134 to 608 amino acids). Over 125 inteins have been cataloged in InBase, the on-line intein database (http://www.neb.com/neb/inteins.html), which includes the Intein Registry[1]. Inteins naturally present in pathogenic microbes represent novel, yet unexploited drug targets. Understanding the chemistry of the splicing reaction has allowed the manipulation of inteins, which are now used in many protein engineering applications[2]
Protein Splicing of the Deinococcus radiodurans Strain R1 Snf2 Intein
Adjacent intein fragments fused to a Snf2/Rad54 helicase-related protein and Snf2/Rad54 helicase were reported for Deinococcus radiodurans R1, leading to the speculation that a frameshift was required for splicing or that trans splicing occurred. However, a type strain (ATCC 13939, RF18410) yielded a single protein that splices by the Ala1 protein splicing pathway, with splicing dependent on adjacent residues
An alternative protein splicing mechanism for inteins lacking an N-terminal nucleophile
Variations in the intein-mediated protein splicing mechanism are becoming more apparent as polymorphisms in conserved catalytic residues are identified. The conserved Ser or Cys at the intein N-terminus and the conserved intein penultimate His are absent in the KlbA family of inteins. These inteins were predicted to be inactive, since an N-terminal Ala cannot perform the initial reaction of the standard protein splicing pathway to yield the requisite N-terminal splice junction (thio)ester. Despite the presence of an N-terminal Ala and a penultimate Ser, the KlbA inteins splice efficiently using an alternative protein splicing mechanism. In this non-canonical pathway, the C-extein nucleophile attacks a peptide bond at the N-terminal splice junction rather than a (thio)ester bond, alleviating the need to form the initial (thio)ester at the N-terminal splice junction. The remainder of the two pathways is the same: branch resolution by Asn cyclization is followed by an acyl rearrangement to form a native peptide bond between the ligated exteins
Splicing of the Mycobacteriophage Bethlehem DnaB Intein: IDENTIFICATION OF A NEW MECHANISTIC CLASS OF INTEINS THAT CONTAIN AN OBLIGATE BLOCK F NUCLEOPHILE*♦
Inteins are single turnover enzymes that splice out of protein precursors during maturation of the host protein (extein). The Cys or Ser at the N terminus of most inteins initiates a four-step protein splicing reaction by forming a (thio)ester bond at the N-terminal splice junction. Several recently identified inteins cannot perform this acyl rearrangement because they do not begin with Cys, Thr, or Ser. This study analyzes one of these, the mycobacteriophage Bethlehem DnaB intein, which we describe here as the prototype for a new class of inteins based on sequence comparisons, reactivity, and mechanism. These Class 3 inteins are characterized by a non-nucleophilic N-terminal residue that co-varies with a non-contiguous Trp, Cys, Thr triplet (WCT) and a Thr or Ser as the first C-extein residue. Several mechanistic differences were observed when compared with standard inteins or previously studied atypical KlbA Ala1 inteins: (a) cleavage at the N-terminal splice junction in the absence of all standard N- and C-terminal splice junction nucleophiles, (b) activation of the N-terminal splice junction by a variant Block B motif that includes the WCT triplet Trp, (c) decay of the branched intermediate by thiols or Cys despite an ester linkage at the C-extein branch point, and (d) an absolute requirement for the WCT triplet Block F Cys. Based on biochemical data and confirmed by molecular modeling, we propose roles for these newly identified conserved residues, a novel protein splicing mechanism that includes a second branched intermediate, and an intein classification with three mechanistic categories
Branched Intermediate Formation Is the Slowest Step in the Protein Splicing Reaction of the Ala1 KlbA Intein from <i>Methanococcus jannaschii</i>
We report the first detailed investigation of the kinetics
of protein
splicing by the <i>Methanococcus jannaschii</i> KlbA (<i>Mja</i> KlbA) intein. This intein has an N-terminal Ala in place
of the nucleophilic Cys or Ser residue that normally initiates splicing
but nevertheless splices efficiently in vivo [Southworth, M. W., Benner,
J., and Perler, F. B. (2000) <i>EMBO J.</i> <i>19</i>, 5019–5026]. To date, the spontaneous nature of the cis splicing
reaction has hindered its examination in vitro. For this reason, we
constructed an <i>Mja</i> KlbA intein–mini-extein
precursor using intein-mediated protein ligation and engineered a
disulfide redox switch that permits initiation of the splicing reaction
by the addition of a reducing agent such as dithiothreitol (DTT).
A fluorescent tag at the C-terminus of the C-extein permits monitoring
of the progress of the reaction. Kinetic analysis of the splicing
reaction of the wild-type precursor (with no substitutions in known
nucleophiles or assisting groups) at various DTT concentrations shows
that formation of the branched intermediate from the precursor is
reversible (forward rate constant of 1.5 × 10<sup>–3</sup> s<sup>–1</sup> and reverse rate constant of 1.7 × 10<sup>–5</sup> s<sup>–1</sup> at 42 °C), whereas the
productive decay of this intermediate to form the ligated exteins
is faster and occurs with a rate constant of 2.2 × 10<sup>–3</sup> s<sup>–1</sup>. This finding conflicts with reports about
standard inteins, for which Asn cyclization has been assigned as the
rate-determining step of the splicing reaction. Despite being the
slowest step of the reaction, branched intermediate formation in the <i>Mja</i> KlbA intein is efficient in comparison with those of
other intein systems. Interestingly, it also appears that this intermediate
is protected against thiolysis by DTT, in contrast to other inteins.
Evidence is presented in support of a tight coupling between the N-terminal
and C-terminal cleavage steps, despite the fact that the C-terminal
single-cleavage reaction occurs in variant <i>Mja</i> KlbA
inteins in the absence of N-terminal cleavage. We posit that the splicing
events in the <i>Mja</i> KlbA system are tightly coordinated
by a network of intra- and interdomain noncovalent interactions, rendering
its function particularly sensitive to minor disruptions in the intein
or extein environments