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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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Comparison of conserved intein sequences. Intein splicing domains contain four conserved motifs consisting of 51 positions in this sequence logo representation (6) of the amino acids that populate these motifs in the three mechanistic classes of inteins. Class 3 inteins are described in this report as lacking the standard N-terminal Ser or Cys nucleophile and containing the WCT triplet. Class 2 inteins consist of KlbA-like inteins, and Class 1 inteins are the standard inteins, which comprise the largest class of inteins. The first 37 sequences in supplemental Table 1 minus the Arsp-FB24 DnaB intein (WDT triplet) were used to generate the sequence logo for Class 3, the five KlbA inteins were used to generate the sequence logo for Class 2, and the remaining 408 inteins present in the June 25, 2009 version of InBase (5) were used in Class 1. Block F, position 8 in Class 3 and positions 8 and 9 in Class 2 represent gaps in this motif due to a variable sized loop. The absence of visible residues in other positions in the Class 2 sequence logo is due to a high degree of variability in these positions and the small class size (five inteins). Residues are colored according to physico-chemical groups: red for acidic, blue for basic, black for hydrophobic, orange for aromatic, cyan for Asn/Gln, and green for Thr/Cys/Ser. The red asterisks mark the positions of the WCT triplet, and the black asterisks mark the position of the conserved Thr and His in Block B. Block A is sometimes referred to as N1, B as N3, F as C2, and G as C1 (6–8).
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Figure 3: Comparison of conserved intein sequences. Intein splicing domains contain four conserved motifs consisting of 51 positions in this sequence logo representation (6) of the amino acids that populate these motifs in the three mechanistic classes of inteins. Class 3 inteins are described in this report as lacking the standard N-terminal Ser or Cys nucleophile and containing the WCT triplet. Class 2 inteins consist of KlbA-like inteins, and Class 1 inteins are the standard inteins, which comprise the largest class of inteins. The first 37 sequences in supplemental Table 1 minus the Arsp-FB24 DnaB intein (WDT triplet) were used to generate the sequence logo for Class 3, the five KlbA inteins were used to generate the sequence logo for Class 2, and the remaining 408 inteins present in the June 25, 2009 version of InBase (5) were used in Class 1. Block F, position 8 in Class 3 and positions 8 and 9 in Class 2 represent gaps in this motif due to a variable sized loop. The absence of visible residues in other positions in the Class 2 sequence logo is due to a high degree of variability in these positions and the small class size (five inteins). Residues are colored according to physico-chemical groups: red for acidic, blue for basic, black for hydrophobic, orange for aromatic, cyan for Asn/Gln, and green for Thr/Cys/Ser. The red asterisks mark the positions of the WCT triplet, and the black asterisks mark the position of the conserved Thr and His in Block B. Block A is sometimes referred to as N1, B as N3, F as C2, and G as C1 (6–8).

Mentions: Over 450 inteins have been identified in archaea, bacteria, viruses, and unicellular eukaryotes (see InBase, the on-line intein data base) (5). Intein splicing domains contain four sequence motifs (see Fig. 3) that are not well conserved in their entirety but contain certain well conserved residues that form part of the protein splicing catalytic center and the hydrophobic core of the splicing domain. There are two concurrently used nomenclatures for these motifs: A or N1, B or N3, F or C2, and G or C1 (6–8). Block A begins at position 1 of the intein and contains the intein N-terminal nucleophile (Ser1 or Cys1). Thr1 has not been observed in inteins identified to date (5) but can perform the same acyl rearrangement as Ser or Cys and has been shown to allow splicing in the S1T mutant of the Thermococcus litoralis DNA pol-2 intein (9). Block B (N3) is usually 60–100 amino acids from the intein N terminus and contains a conserved Thr at position 7 and a His at position 10, both of which assist reactions at the N-terminal splice junction (1, 2, 6–8). Residues in Block F (C2), especially an Asp or a Trp in position 4, have been shown to be essential for N-terminal cleavage, leading many authors to conclude that Block F may be important in orienting the C-extein nucleophile for attack on the N-terminal scissile bond and/or may participate in catalysis at the N-terminal splice site (10–12). In the Methanococcus jannaschii (Mja) KlbA intein, Asp147 likely assists catalysis by helping to activate the Cys+1 nucleophile (12). Block G (C1) includes the last 7 amino acids of the intein and the first residue of the C-extein (termed the +1 amino acid). The intein penultimate His usually assists Asn cyclization, although some inteins use other residues for this purpose (1, 2, 12). Block F is generally <10 amino acids from Block G.


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)

Comparison of conserved intein sequences. Intein splicing domains contain four conserved motifs consisting of 51 positions in this sequence logo representation (6) of the amino acids that populate these motifs in the three mechanistic classes of inteins. Class 3 inteins are described in this report as lacking the standard N-terminal Ser or Cys nucleophile and containing the WCT triplet. Class 2 inteins consist of KlbA-like inteins, and Class 1 inteins are the standard inteins, which comprise the largest class of inteins. The first 37 sequences in supplemental Table 1 minus the Arsp-FB24 DnaB intein (WDT triplet) were used to generate the sequence logo for Class 3, the five KlbA inteins were used to generate the sequence logo for Class 2, and the remaining 408 inteins present in the June 25, 2009 version of InBase (5) were used in Class 1. Block F, position 8 in Class 3 and positions 8 and 9 in Class 2 represent gaps in this motif due to a variable sized loop. The absence of visible residues in other positions in the Class 2 sequence logo is due to a high degree of variability in these positions and the small class size (five inteins). Residues are colored according to physico-chemical groups: red for acidic, blue for basic, black for hydrophobic, orange for aromatic, cyan for Asn/Gln, and green for Thr/Cys/Ser. The red asterisks mark the positions of the WCT triplet, and the black asterisks mark the position of the conserved Thr and His in Block B. Block A is sometimes referred to as N1, B as N3, F as C2, and G as C1 (6–8).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Comparison of conserved intein sequences. Intein splicing domains contain four conserved motifs consisting of 51 positions in this sequence logo representation (6) of the amino acids that populate these motifs in the three mechanistic classes of inteins. Class 3 inteins are described in this report as lacking the standard N-terminal Ser or Cys nucleophile and containing the WCT triplet. Class 2 inteins consist of KlbA-like inteins, and Class 1 inteins are the standard inteins, which comprise the largest class of inteins. The first 37 sequences in supplemental Table 1 minus the Arsp-FB24 DnaB intein (WDT triplet) were used to generate the sequence logo for Class 3, the five KlbA inteins were used to generate the sequence logo for Class 2, and the remaining 408 inteins present in the June 25, 2009 version of InBase (5) were used in Class 1. Block F, position 8 in Class 3 and positions 8 and 9 in Class 2 represent gaps in this motif due to a variable sized loop. The absence of visible residues in other positions in the Class 2 sequence logo is due to a high degree of variability in these positions and the small class size (five inteins). Residues are colored according to physico-chemical groups: red for acidic, blue for basic, black for hydrophobic, orange for aromatic, cyan for Asn/Gln, and green for Thr/Cys/Ser. The red asterisks mark the positions of the WCT triplet, and the black asterisks mark the position of the conserved Thr and His in Block B. Block A is sometimes referred to as N1, B as N3, F as C2, and G as C1 (6–8).
Mentions: Over 450 inteins have been identified in archaea, bacteria, viruses, and unicellular eukaryotes (see InBase, the on-line intein data base) (5). Intein splicing domains contain four sequence motifs (see Fig. 3) that are not well conserved in their entirety but contain certain well conserved residues that form part of the protein splicing catalytic center and the hydrophobic core of the splicing domain. There are two concurrently used nomenclatures for these motifs: A or N1, B or N3, F or C2, and G or C1 (6–8). Block A begins at position 1 of the intein and contains the intein N-terminal nucleophile (Ser1 or Cys1). Thr1 has not been observed in inteins identified to date (5) but can perform the same acyl rearrangement as Ser or Cys and has been shown to allow splicing in the S1T mutant of the Thermococcus litoralis DNA pol-2 intein (9). Block B (N3) is usually 60–100 amino acids from the intein N terminus and contains a conserved Thr at position 7 and a His at position 10, both of which assist reactions at the N-terminal splice junction (1, 2, 6–8). Residues in Block F (C2), especially an Asp or a Trp in position 4, have been shown to be essential for N-terminal cleavage, leading many authors to conclude that Block F may be important in orienting the C-extein nucleophile for attack on the N-terminal scissile bond and/or may participate in catalysis at the N-terminal splice site (10–12). In the Methanococcus jannaschii (Mja) KlbA intein, Asp147 likely assists catalysis by helping to activate the Cys+1 nucleophile (12). Block G (C1) includes the last 7 amino acids of the intein and the first residue of the C-extein (termed the +1 amino acid). The intein penultimate His usually assists Asn cyclization, although some inteins use other residues for this purpose (1, 2, 12). Block F is generally <10 amino acids from Block G.

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