Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp¹²² and Asp¹⁹³ are crucial to the double‐stranded DNA cleavage activity whereas Asp²¹⁸ is not

Abstract

Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their active center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the activesite residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp 122 (in Block C) and Asp 193 (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp 218 (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PIMleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp 122 and Additional Supporting Information may be found in the online version of this article.Abbreviations: 2-AP, 2 aminopurine; bp, base pair; BPB, bromophenol blue; EDTA, ethylene diamine tetraacetic acid; form I DNA, negatively supercoiled DNA; form II DNA, nicked circular DNA; form III DNA, linear double-stranded DNA; ODN, oligonucleotide; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PI-MleI, M. leprae RecA intein; SDS, sodium dodecyl sulfate. Asp 193 in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.

Protein Splicing Activity of the Haloferax volcanii PolB-c Intein Is Sensitive to Homing Endonuclease Domain Mutations

“…We made an alignment with eight other haloarchaea that have a PolB-c intein to guide the selection of nonsynonymous substitutions (Figure S1). We chose these residues as they are highly conserved among the nine inteins but should not affect DNA and metal cofactor binding as much as altering the active site Asp residues. ,− We also were concerned about mutating negatively charged residues, which can be important for protein stability in halophilic proteins in high salt . We elected to change intein residue Leu211 to Gly, Tyr214 to Ala, and F330 to Ala, in accordance with H. volcanii codon usage, and used the typical intein numbering scheme with the first intein residue as residue 1. , Comparing these positions to the equivalent positions in the crystal structure of the PI-SceI homing endonuclease (PDB code 1VDE), L211 and Y214 would be part of the α helix at the motif interface but likely point away from the interface (residues L212 and W215 in the PI-SceI structure), whereas F330 would be part of the opposing helix but likely point away from the motif interface (residue F318 in PI-SceI) …”

A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein

“…Due to its primary structure including a partially degraded and probably inactive endonuclease domain, this intein can be assigned to the proposed homing cycle of inteins at the stage Bintein with no or non-functional endonuclease^and has already undergone a process of degradation in the course of evolution (Gogarten et al 2002). In addition to the reduced size of the endonuclease, acidic amino acids that are relevant for activity are also completely missing in Pto VMA intein (Singh et al 2010). A functional expression and assay system may provide an important tool to experimentally remove or mutate the endonuclease domain in a stepwise manner by molecular biology techniques.…”