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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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Related in: MedlinePlus

Three intein classes splice by different mechanisms. Most inteins follow the standard protein splicing pathway (Class 1). However, some inteins splice using modifications of this mechanism. The Class 1 standard protein splicing pathway consists of four nucleophilic displacement reactions: Step 1, an N-(S/O) acyl shift at the N-terminal splice junction; Step 2, transesterification to form the Block G branched intermediate; Step 3, Asn cyclization to cleave the C-terminal splice junction; Step 4, a spontaneous (S/O)-N acyl shift to form a native peptide bond between the exteins; and Step 5, the slow hydrolysis of the succinimide ring. Although Thr could perform the initial acyl shift, it has not been observed in standard inteins (5, 9). Class 2 and Class 3 inteins lack an N-terminal Ser or Cys, so they cannot perform the acyl shift that initiates the splicing reaction in Class 1 inteins. In Class 2 inteins, Cys+1 directly attacks an amide bond at the N-terminal splice junction to form the standard Block G branched intermediate (III). Known examples of Class 2 inteins are the KlbA inteins. The Class 3 protein splicing pathway also consists of four nucleophilic displacement reactions, including two branched intermediates. In Step 1, the Cys in Block F (Cys320 in the MP-Be DnaB intein) attacks the peptide bond at the N-terminal splice junction, forming the Block F branched intermediate (VIII), which is thiol labile. In Step 2, the N-extein is transferred from the side chain of the Block F Cys to the side chain of the +1 residue (Thr+1 in the MP-Be DnaB intein) by a transesterification reaction resulting in the formation of the same Block G branched intermediate (III) as in Class 1; this is an ester linkage in the MP-Be DnaB intein and is proposed to be resistant to cleavage by thiols at room temperature. Once the Block G branched intermediate (III) is formed, the remainder of the splicing pathway is the same in all inteins. Class 3 inteins contain the WCT triplet, whereas Class 2 inteins do not. Residues within the intein assist these reactions. Tetrahedral intermediates are not shown. X represents the sulfur or oxygen atom in the side chain of Ser, Thr, or Cys.
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Figure 1: Three intein classes splice by different mechanisms. Most inteins follow the standard protein splicing pathway (Class 1). However, some inteins splice using modifications of this mechanism. The Class 1 standard protein splicing pathway consists of four nucleophilic displacement reactions: Step 1, an N-(S/O) acyl shift at the N-terminal splice junction; Step 2, transesterification to form the Block G branched intermediate; Step 3, Asn cyclization to cleave the C-terminal splice junction; Step 4, a spontaneous (S/O)-N acyl shift to form a native peptide bond between the exteins; and Step 5, the slow hydrolysis of the succinimide ring. Although Thr could perform the initial acyl shift, it has not been observed in standard inteins (5, 9). Class 2 and Class 3 inteins lack an N-terminal Ser or Cys, so they cannot perform the acyl shift that initiates the splicing reaction in Class 1 inteins. In Class 2 inteins, Cys+1 directly attacks an amide bond at the N-terminal splice junction to form the standard Block G branched intermediate (III). Known examples of Class 2 inteins are the KlbA inteins. The Class 3 protein splicing pathway also consists of four nucleophilic displacement reactions, including two branched intermediates. In Step 1, the Cys in Block F (Cys320 in the MP-Be DnaB intein) attacks the peptide bond at the N-terminal splice junction, forming the Block F branched intermediate (VIII), which is thiol labile. In Step 2, the N-extein is transferred from the side chain of the Block F Cys to the side chain of the +1 residue (Thr+1 in the MP-Be DnaB intein) by a transesterification reaction resulting in the formation of the same Block G branched intermediate (III) as in Class 1; this is an ester linkage in the MP-Be DnaB intein and is proposed to be resistant to cleavage by thiols at room temperature. Once the Block G branched intermediate (III) is formed, the remainder of the splicing pathway is the same in all inteins. Class 3 inteins contain the WCT triplet, whereas Class 2 inteins do not. Residues within the intein assist these reactions. Tetrahedral intermediates are not shown. X represents the sulfur or oxygen atom in the side chain of Ser, Thr, or Cys.

Mentions: Inteins are the protein equivalent of introns. These intervening sequences must be post-translationally removed from the surrounding host protein fragments (exteins) before the host protein becomes functional. The intein together with the first C-extein amino acid act as a single turnover enzyme that removes the intein from the precursor protein and seamlessly joins the exteins. No external cofactors or energy sources are required for this self-catalytic protein splicing reaction. The standard intein-mediated protein splicing pathway consists of four coordinated nucleophilic displacements (Fig. 1) and has been extensively reviewed (1, 2). The first step is an acyl rearrangement in which the first residue of the intein (Cys1 or Ser1) reacts with the carbonyl carbon of the preceding extein residue, resulting in a (thio)ester bond at the N-terminal splice junction (II). The first C-extein residue (Cys+1, Ser+1, or Thr+1) then attacks this (thio)ester bond, cleaving the N-terminal splice junction and transferring the N-extein to its side chain to form the Block G branched (thio)ester intermediate (III). The Block G branched intermediate (BI)3 is resolved by cyclization of the intein C-terminal Asn, which results in cleavage of the C-terminal splice junction and release of the intein (IV) from the (thio)ester linked exteins (V). A native peptide bond is formed between the exteins by a spontaneous acyl shift (VI). The succinimide at the end of the intein (IV) eventually hydrolyzes to form Asn (VII) or isoasparagine.


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)

Three intein classes splice by different mechanisms. Most inteins follow the standard protein splicing pathway (Class 1). However, some inteins splice using modifications of this mechanism. The Class 1 standard protein splicing pathway consists of four nucleophilic displacement reactions: Step 1, an N-(S/O) acyl shift at the N-terminal splice junction; Step 2, transesterification to form the Block G branched intermediate; Step 3, Asn cyclization to cleave the C-terminal splice junction; Step 4, a spontaneous (S/O)-N acyl shift to form a native peptide bond between the exteins; and Step 5, the slow hydrolysis of the succinimide ring. Although Thr could perform the initial acyl shift, it has not been observed in standard inteins (5, 9). Class 2 and Class 3 inteins lack an N-terminal Ser or Cys, so they cannot perform the acyl shift that initiates the splicing reaction in Class 1 inteins. In Class 2 inteins, Cys+1 directly attacks an amide bond at the N-terminal splice junction to form the standard Block G branched intermediate (III). Known examples of Class 2 inteins are the KlbA inteins. The Class 3 protein splicing pathway also consists of four nucleophilic displacement reactions, including two branched intermediates. In Step 1, the Cys in Block F (Cys320 in the MP-Be DnaB intein) attacks the peptide bond at the N-terminal splice junction, forming the Block F branched intermediate (VIII), which is thiol labile. In Step 2, the N-extein is transferred from the side chain of the Block F Cys to the side chain of the +1 residue (Thr+1 in the MP-Be DnaB intein) by a transesterification reaction resulting in the formation of the same Block G branched intermediate (III) as in Class 1; this is an ester linkage in the MP-Be DnaB intein and is proposed to be resistant to cleavage by thiols at room temperature. Once the Block G branched intermediate (III) is formed, the remainder of the splicing pathway is the same in all inteins. Class 3 inteins contain the WCT triplet, whereas Class 2 inteins do not. Residues within the intein assist these reactions. Tetrahedral intermediates are not shown. X represents the sulfur or oxygen atom in the side chain of Ser, Thr, or Cys.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Three intein classes splice by different mechanisms. Most inteins follow the standard protein splicing pathway (Class 1). However, some inteins splice using modifications of this mechanism. The Class 1 standard protein splicing pathway consists of four nucleophilic displacement reactions: Step 1, an N-(S/O) acyl shift at the N-terminal splice junction; Step 2, transesterification to form the Block G branched intermediate; Step 3, Asn cyclization to cleave the C-terminal splice junction; Step 4, a spontaneous (S/O)-N acyl shift to form a native peptide bond between the exteins; and Step 5, the slow hydrolysis of the succinimide ring. Although Thr could perform the initial acyl shift, it has not been observed in standard inteins (5, 9). Class 2 and Class 3 inteins lack an N-terminal Ser or Cys, so they cannot perform the acyl shift that initiates the splicing reaction in Class 1 inteins. In Class 2 inteins, Cys+1 directly attacks an amide bond at the N-terminal splice junction to form the standard Block G branched intermediate (III). Known examples of Class 2 inteins are the KlbA inteins. The Class 3 protein splicing pathway also consists of four nucleophilic displacement reactions, including two branched intermediates. In Step 1, the Cys in Block F (Cys320 in the MP-Be DnaB intein) attacks the peptide bond at the N-terminal splice junction, forming the Block F branched intermediate (VIII), which is thiol labile. In Step 2, the N-extein is transferred from the side chain of the Block F Cys to the side chain of the +1 residue (Thr+1 in the MP-Be DnaB intein) by a transesterification reaction resulting in the formation of the same Block G branched intermediate (III) as in Class 1; this is an ester linkage in the MP-Be DnaB intein and is proposed to be resistant to cleavage by thiols at room temperature. Once the Block G branched intermediate (III) is formed, the remainder of the splicing pathway is the same in all inteins. Class 3 inteins contain the WCT triplet, whereas Class 2 inteins do not. Residues within the intein assist these reactions. Tetrahedral intermediates are not shown. X represents the sulfur or oxygen atom in the side chain of Ser, Thr, or Cys.
Mentions: Inteins are the protein equivalent of introns. These intervening sequences must be post-translationally removed from the surrounding host protein fragments (exteins) before the host protein becomes functional. The intein together with the first C-extein amino acid act as a single turnover enzyme that removes the intein from the precursor protein and seamlessly joins the exteins. No external cofactors or energy sources are required for this self-catalytic protein splicing reaction. The standard intein-mediated protein splicing pathway consists of four coordinated nucleophilic displacements (Fig. 1) and has been extensively reviewed (1, 2). The first step is an acyl rearrangement in which the first residue of the intein (Cys1 or Ser1) reacts with the carbonyl carbon of the preceding extein residue, resulting in a (thio)ester bond at the N-terminal splice junction (II). The first C-extein residue (Cys+1, Ser+1, or Thr+1) then attacks this (thio)ester bond, cleaving the N-terminal splice junction and transferring the N-extein to its side chain to form the Block G branched (thio)ester intermediate (III). The Block G branched intermediate (BI)3 is resolved by cyclization of the intein C-terminal Asn, which results in cleavage of the C-terminal splice junction and release of the intein (IV) from the (thio)ester linked exteins (V). A native peptide bond is formed between the exteins by a spontaneous acyl shift (VI). The succinimide at the end of the intein (IV) eventually hydrolyzes to form Asn (VII) or isoasparagine.

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