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Splicing of the mycobacteriophage Bethlehem DnaB intein: identification of a new mechanistic class of inteins that contain an obligate block F nucleophile.

Tori K, Dassa B, Johnson MA, Southworth MW, Brace LE, Ishino Y, Pietrokovski S, Perler FB - J. Biol. Chem. (2009)

Bottom Line: Several recently identified inteins cannot perform this acyl rearrangement because they do not begin with Cys, Thr, or Ser.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.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.

View Article: PubMed Central - PubMed

Affiliation: New England Biolabs, Ipswich, Massachusetts 01938, USA.

ABSTRACT
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 Ala(1) 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.

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A schematic representation of splicing and off-pathway reactions. The MP-Be DnaB intein (I) was cloned into a model precursor consisting of MBP as the N-extein (M) and a fragment of paramyosin (P) as the C-extein, along with 5 native extein residues flanking each splice site. Protein splicing (S) yields MP and free intein, whereas off-pathway N-terminal splice junction cleavage (N) yields M + IP and off-pathway C-terminal splice junction cleavage (C) yields C-terminal cleavage products (MI + P). Cleavage of the (thio)ester linkage in branched intermediates also yields M + IP. Some mutations result in double cleavage at both splice junctions to yield M + I + P. Molecular weights of MP-Be DnaB intein MIP precursor and products are listed.
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Figure 2: A schematic representation of splicing and off-pathway reactions. The MP-Be DnaB intein (I) was cloned into a model precursor consisting of MBP as the N-extein (M) and a fragment of paramyosin (P) as the C-extein, along with 5 native extein residues flanking each splice site. Protein splicing (S) yields MP and free intein, whereas off-pathway N-terminal splice junction cleavage (N) yields M + IP and off-pathway C-terminal splice junction cleavage (C) yields C-terminal cleavage products (MI + P). Cleavage of the (thio)ester linkage in branched intermediates also yields M + IP. Some mutations result in double cleavage at both splice junctions to yield M + I + P. Molecular weights of MP-Be DnaB intein MIP precursor and products are listed.

Mentions: The catalytic pathway in all inteins is highly coordinated, but the precise mechanism that controls this series of reactions remains unclear. The mechanism may also vary among different inteins. For example, some inteins display coupling of N-terminal and C-terminal reactions (3), whereas others do not (4). Intein mutations and/or the presence of artificial, non-native exteins often disrupt the coordination of these steps, yielding off-pathway single or double splice junction cleavage products that are incapable of forming the mature host protein (Fig. 2). Off-pathway C-terminal splice junction cleavage is caused by Asn cyclization in precursors (I or II) prior to BI (III) formation, whereas off-pathway N-terminal splice junction cleavage is caused by cleavage of the (thio)ester bond in the linear (II) or branched (III) (thio)ester intermediates. Off-pathway splice junction cleavage can result from either an increase in the rate of cleavage at that splice junction or a decrease in the reaction rate of another step. However, to our knowledge, neither splicing nor off-pathway cleavage of an amide bond at the N-terminal splice junction has been observed in standard inteins after non-conservative substitution of Ser1 or Cys1.


Splicing of the mycobacteriophage Bethlehem DnaB intein: identification of a new mechanistic class of inteins that contain an obligate block F nucleophile.

Tori K, Dassa B, Johnson MA, Southworth MW, Brace LE, Ishino Y, Pietrokovski S, Perler FB - J. Biol. Chem. (2009)

A schematic representation of splicing and off-pathway reactions. The MP-Be DnaB intein (I) was cloned into a model precursor consisting of MBP as the N-extein (M) and a fragment of paramyosin (P) as the C-extein, along with 5 native extein residues flanking each splice site. Protein splicing (S) yields MP and free intein, whereas off-pathway N-terminal splice junction cleavage (N) yields M + IP and off-pathway C-terminal splice junction cleavage (C) yields C-terminal cleavage products (MI + P). Cleavage of the (thio)ester linkage in branched intermediates also yields M + IP. Some mutations result in double cleavage at both splice junctions to yield M + I + P. Molecular weights of MP-Be DnaB intein MIP precursor and products are listed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2807308&req=5

Figure 2: A schematic representation of splicing and off-pathway reactions. The MP-Be DnaB intein (I) was cloned into a model precursor consisting of MBP as the N-extein (M) and a fragment of paramyosin (P) as the C-extein, along with 5 native extein residues flanking each splice site. Protein splicing (S) yields MP and free intein, whereas off-pathway N-terminal splice junction cleavage (N) yields M + IP and off-pathway C-terminal splice junction cleavage (C) yields C-terminal cleavage products (MI + P). Cleavage of the (thio)ester linkage in branched intermediates also yields M + IP. Some mutations result in double cleavage at both splice junctions to yield M + I + P. Molecular weights of MP-Be DnaB intein MIP precursor and products are listed.
Mentions: The catalytic pathway in all inteins is highly coordinated, but the precise mechanism that controls this series of reactions remains unclear. The mechanism may also vary among different inteins. For example, some inteins display coupling of N-terminal and C-terminal reactions (3), whereas others do not (4). Intein mutations and/or the presence of artificial, non-native exteins often disrupt the coordination of these steps, yielding off-pathway single or double splice junction cleavage products that are incapable of forming the mature host protein (Fig. 2). Off-pathway C-terminal splice junction cleavage is caused by Asn cyclization in precursors (I or II) prior to BI (III) formation, whereas off-pathway N-terminal splice junction cleavage is caused by cleavage of the (thio)ester bond in the linear (II) or branched (III) (thio)ester intermediates. Off-pathway splice junction cleavage can result from either an increase in the rate of cleavage at that splice junction or a decrease in the reaction rate of another step. However, to our knowledge, neither splicing nor off-pathway cleavage of an amide bond at the N-terminal splice junction has been observed in standard inteins after non-conservative substitution of Ser1 or Cys1.

Bottom Line: Several recently identified inteins cannot perform this acyl rearrangement because they do not begin with Cys, Thr, or Ser.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.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.

View Article: PubMed Central - PubMed

Affiliation: New England Biolabs, Ipswich, Massachusetts 01938, USA.

ABSTRACT
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 Ala(1) 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.

Show MeSH
Related in: MedlinePlus