Cofactor analogue-induced chemical reactivation of endonuclease activity in a DNA cleavage/methylation deficient TspGWI N₄₇₃A variant in the NPPY motif.
We reported previously that TspGWI, a prototype enzyme of a new Thermus sp. family of restriction endonucleases-methyltransferases (REases-MTases), undergoes the novel phenomenon of sinefungin (SIN)-caused specificity transition. Here we investigated mutant TspGWI N473A, containing a single amino acid (aa) substitution in the NPPY motif of the MTase. Even though the aa substitution is located within the MTase polypeptide segment, DNA cleavage and modification are almost completely abolished, indicating that the REase and MTase are intertwined. Remarkably, the TspGWI N473A REase functionality can be completely reconstituted by the addition of SIN. We hypothesize that SIN binds specifically to the enzyme and restores the DNA cleavage-competent protein tertiary structure. This indicates the significant role of allosteric effectors in DNA cleavage in Thermus sp. enzymes. This is the first case of REase mutation suppression by an S-adenosylmethionine (SAM) cofactor analogue. Moreover, the TspGWI N473A clone strongly affects E. coli division control, acting as a 'selfish gene'. The mutant lacks the competing MTase activity and therefore might be useful for applications in DNA manipulation. Here we present a case study of a novel strategy for REase activity/specificity alteration by a single aa substitution, based on the bioinformatic analysis of active motif locations, combining (a) aa sequence engineering (b) the alteration of protein enzymatic properties, and (c) the use of cofactor-analogue cleavage reconstitution and stimulation.
Department of Molecular Biotechnology, Institute for Environmental and Human Health Protection, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-952, Gdansk, Poland, a.zylicz-stachula@ug.edu.pl.
Agnieszka
A
Zylicz-Stachula
Joanna
J
Jeżewska-Frąckowiak
Piotr M
PM
Skowron
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D053837:DNA Cleavage; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D008780:Methyltransferases; D004262:DNA Restriction Enzymes