Crystallographic and Mutational Studies of Mycobacterium tuberculosis recA Mini-inteins Suggest a Pivotal Role for a Highly Conserved Aspartate Residue

Roey, P. Van; Pereira, Brian J.G.; Li, Zhong; Hiraga, Kaori; Belfort, Marlene; Derbyshire, Victoria

doi:10.1016/j.jmb.2006.12.050

Cited by 74 publications

(160 citation statements)

References 41 publications

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“…The essential characteristics of class 3 inteins are (i) cleavage at the N-terminal splice junction in the absence of all class 1 and class 2 splice junction nucleophiles at A:1, G:7, and G:8; (ii) activation of the N-terminal splice junction by a block B motif that includes the conserved His (2,20). Class 1 and 2 inteins also have a hydrophobic residue at the B:12 position (8,11,21), including Trp, which suggests that this position is involved in hydrophobic packing in all classes of inteins.…”

Section: Discussionmentioning

confidence: 99%

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Expanding the Definition of Class 3 Inteins and Their Proposed Phage Origin

Tori

Perler

2011

J Bacteriol

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This study determined which class the mycobacteriophage Catera Gp206 and Nocardioides sp. JS614 TOPRIM inteins belong to based on catalytic mechanism. The mycobacteriophage Catera Gp206 intein (starting with Ser 1 ) is a class 3 intein, and its Ser 1 residue is not required for splicing. Based on phylogenetic analysis, we propose that class 3 inteins arose from a single mutated intein that was spread by phage into predominantly helicase genes in various phages and their hosts.

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Section: Discussionmentioning

confidence: 99%

“…3). In class 1 and 2 inteins, mutations at F:4 inhibit splicing because this residue assists reactions at one or both splice junctions, depending on the individual intein (2,8,13,(19)(20)(21). In class 3 inteins, Ala substitution for this residue blocks all N-terminal cleavage reactions, including BI formation.…”

Section: Splicing Of Wild-type Inteinsmentioning

confidence: 99%

Expanding the Definition of Class 3 Inteins and Their Proposed Phage Origin

Tori

Perler

2011

J Bacteriol

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“…We used the RecA intein as the model in this study because of the wealth of structural and functional information available for this intein (13)(14)(15) and because intein inhibitors isolated against the RecA intein are also active against the DnaB intein (16), which is required for viability. Furthermore, RecA is one of the proteins associated with M. tuberculosis drug resistance (17), and mutation of RecA causes sensitivity to DNA-damaging agents and increased susceptibility to metronidazole (18 It has been reported that the intein activity can be suppressed by Zn 2ϩ and Cu 2ϩ ions (19 -21).…”

mentioning

confidence: 99%

“…Functional studies also indicated that several other conserved residues in the M. tuberculosis RecA intein are required for protein splicing, including His-73 and His-439. Crystal structures suggest that these residues are close in space and form the active site core of the M. tuberculosis RecA intein (13). Because cysteine and histidine residues are favored sites for platinum(II) binding in proteins, we hypothesized that platinum(II) complexes would be particularly suited to intein inhibition.…”

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confidence: 99%

Cisplatin Inhibits Protein Splicing, Suggesting Inteins as Therapeutic Targets in Mycobacteria

Zhang

Zheng

Callahan

et al. 2011

Journal of Biological Chemistry

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Mycobacterium tuberculosis harbors three protein splicing elements, called inteins, in critical genes and their protein products. Post-translational removal of the inteins occurs autocatalytically and is required for function of the respective M. tuberculosis proteins. Inteins are therefore potential targets for antimycobacterial agents. In this work, we report that the splicing activity of the intein present in the RecA recombinase of M. tuberculosis is potently inhibited by the anticancer drug cisplatin (cis-diamminedichloro-platinum(II)). This previously unrecognized activity of cisplatin was established using both an in vitro intein splicing assay, which yielded an IC 50 of ϳ2 M, and a genetic reporter for intein splicing in Escherichia coli. Testing of related platinum(II) complexes indicated that the inhibition activity is highly structure-dependent, with cisplatin exhibiting the best inhibitory effect. Finally, we report that cisplatin is toxic toward M. tuberculosis with a minimum inhibitory concentration of ϳ40 M, and in genetic experiments conducted with the related Mycobacterium bovis bacillus Calmette-Guérrin (BCG) strain, we show that cisplatin toxicity can be mitigated by intein overexpression. We propose that cisplatin inhibits intein activity by modifying at least one conserved cysteine residue that is required for splicing. Together these results identify a novel active site inhibitor of inteins and validate inteins as viable targets for small molecule inhibition in mycobacteria.Tuberculosis remains a leading cause of death worldwide (1, 2), and the global emergence of multidrug-resistant Mycobacterium tuberculosis portends further challenges in TB Several microbial pathogens, including M. tuberculosis, contain protein self-splicing elements called inteins, which interrupt critical genes and their protein products. These inteins are therefore potential novel targets for antibiotic development, particularly because no inteins are present in the human genome. In addition to M. tuberculosis, self-splicing inteins reside in critical proteins of Mycobacterium leprae, Coxiella burnetii, and Cryptococcus neoformans, the etiological agents of leprosy, Q fever, and cryptococcosis, respectively (6 -8). For these pathogens to survive, the inteins must catalyze a multistep reaction in which they are excised from the precursor protein and the two flanking sequences, called exteins, must be ligated to form a functional product protein (9 -11). Because inteins exert control, via splicing, over the function of pathogen-specific proteins, these self-splicing elements are attractive candidates for inhibition studies. The pursuit of intein inhibitors, whether as mechanistic probes or as an avenue for developing antibacterials, does face a unique challenge; unlike a conventional enzyme inhibitor, an effective antagonist of intein splicing must outcompete a substrate that is covalently tethered to the enzyme.M. tuberculosis has inteins in three distinct genes: in the dnaB helicase gene, in the recA recombinase gene,...

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“…Even if a branched ester can form, as we believe we observed with the intein with a single Cys320Ala mutation, uncoupled C-terminal cleavage is still prevented. Perhaps Cys320 is required to properly coordinate the active site to promote Asn cyclization, as with the Asp at that position in the Mycobacterium tuberculosis RecA intein (15,16). Alternatively, the Cys320 could promote proper branched-ester formation, with Asn cyclization being coupled to splicing in the context of a branched ester, as with the Mycobacterium xenopi GyrA intein (4).…”

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confidence: 99%

Canonical Protein Splicing of a Class 1 Intein That Has a Class 3 Noncanonical Sequence Motif

Reitter

Mills

2011

J Bacteriol

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A Thermobifida fusca intein has two characteristics of class 3 inteins: a noncontiguous covariant Trp-Cys-Thr triplet and a Ser flanking its C terminus. However, it has Cys at position one, characteristic of class 1 inteins. Splicing does not require the internal Cys, which may instead coordinate the active site. Therefore, the intein is class 1.Protein splicing is a posttranslational process by which an intervening polypeptide, or intein, is responsible for its own excision from the flanking polypeptides, or exteins, concomitant with extein ligation (9, 10).The canonical mechanism of protein splicing has four steps ( Fig. 1) (9, 10). First, the peptide bond linking the N-terminal extein (N extein) to the intein is converted to a thioester or ester by nucleophilic attack by the side chain of the first intein residue, Cys or Ser. Second, the first residue of the C-terminal extein (C extein) serves as a nucleophile to attack the nascent ester, resulting in transfer of the N extein from the side chain of the first intein residue to the side chain of the first C-extein residue. Third, the conserved C-terminal Asn of the intein cyclizes, and this is coupled to cleavage of the peptide bond linking the intein to the exteins. Finally, the ester linking the exteins converts to an amide and the intein C-terminal aminosuccinimide may be hydrolyzed.Two classes of inteins that lack an N-terminal nucleophile and can promote splicing without the canonical first step have been described previously ( Fig. 1) (2, 14). Class 2 inteins can bypass the first step of splicing; splicing is initiated by attack of the downstream nucleophile on the N-terminal splice junction (13,14). Class 3 inteins lack an N-terminal nucleophile and have a covariant Trp-Cys-Thr conserved triplet of noncontiguous residues. For the Mycobacterium phage Bethlehem DnaB and Deinococcus radiodurans Snf2 inteins, the Cys of the covariant triplet is responsible for initial attack on the N-terminal splice junction peptide bond, creating an internal branched thioester (2, 14). The N extein is then transferred from the side chain of the internal Cys to the side chain of the Ser flanking the intein C terminus. As most class 3 inteins are flanked by Ser or Thr, this mechanism has a compelling chemical logic: the more nucleophilic internal Cys attacks the amide, and the attack of the less nucleophilic downstream Ser is directed to the more electrophilic thioester, creating a more stable oxygen ester and driving the equilibrium of step two toward splicing.

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Crystallographic and Mutational Studies of Mycobacterium tuberculosis recA Mini-inteins Suggest a Pivotal Role for a Highly Conserved Aspartate Residue

Cited by 74 publications

References 41 publications

Expanding the Definition of Class 3 Inteins and Their Proposed Phage Origin

Expanding the Definition of Class 3 Inteins and Their Proposed Phage Origin

Cisplatin Inhibits Protein Splicing, Suggesting Inteins as Therapeutic Targets in Mycobacteria

Canonical Protein Splicing of a Class 1 Intein That Has a Class 3 Noncanonical Sequence Motif

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