Structure-activity relationships in Kluyveromyces lactis γ-toxin, a eukaryal tRNA anticodon nuclease Journal Article

   

Authors:	Keppetipola, N.; Jain, R.; Meineke, B.; Diver, M.; Shuman, S.
Article Title:	Structure-activity relationships in Kluyveromyces lactis γ-toxin, a eukaryal tRNA anticodon nuclease
Abstract:	tRNA anticodon damage inflicted by secreted ribotoxins such as Kluyveromyces lactis γ-toxin and bacterial colicins underlies a rudimentary innate immune system that distinguishes self from nonself species. The intracellular expression of γ-toxin (a 232-amino acid polypeptide) arrests the growth of Saccharomyces cerevisiae by incising a single RNA phosphodiester 3′ of the modified wobble base of tRNA<sup>Glu</sup>. Fungal γ-toxin bears no primary structure similarity to any known nuclease and has no plausible homologs in the protein database. To gain insight to γ-toxin's mechanism, we tested the effects of alanine mutations at 62 basic, acidic, and polar amino acids on ribotoxin activity in vivo. We thereby identified 22 essential residues, including 10 lysines, seven arginines, three glutamates, one cysteine, and one histidine (His209, the only histidine present in γ-toxin). Structure-activity relations were gleaned from the effects of 44 conservative substitutions. Recombinant tag-free γ-toxin, a monomeric protein, incised an oligonucleotide corresponding to the anticodon stem-loop of tRNA<sup>Glu</sup> at a single phosphodiester 3′ of the wobble uridine. The anticodon nuclease was metal independent. RNA cleavage was abolished by ribose 2′-H and 2′-F modifications of the wobble uridine. Mutating His209 to alanine, glutamine, or asparagine abolished nuclease activity. We propose that γ-toxin catalyzes an RNase A-like transesterification reaction that relies on His209 and a second nonhistidine side chain as general acid-base catalysts. Copyright © 2009 RNA Society.
Keywords:	protein expression; unclassified drug; gene mutation; nonhuman; in vivo study; enzyme activity; structure activity relation; structure-activity relationship; bacteria (microorganisms); amino acid sequence; molecular sequence data; saccharomyces cerevisiae; eukaryota; recombinant proteins; substrate specificity; recombinant protein; nuclease; 3' untranslated region; alanine; transfer rna; rna, transfer; glutamic acid; oligonucleotide; genetic conservation; catalyst; lysine; cysteine; fungus growth; general acid-base catalysis; innate immune system; rna damage; arginine; asparagine; gamma toxin; glutamic acid transfer rna; histidine; metal; monomer; mycotoxin; ribonuclease a; uridine; anticodon; enzyme mechanism; eukaryote; kluyveromyces; rna cleavage; transesterification; killer factors, yeast; ribonucleases; kluyveromyces lactis
Journal Title:	RNA
Volume:	15
Issue:	6
ISSN:	1355-8382
Publisher:	Cold Spring Harbor Laboratory Press
Date Published:	2009-06-01
Start Page:	1036
End Page:	1044
Language:	English
DOI:	10.1261/rna.1637809
PUBMED:	19383764
PROVIDER:	scopus
PMCID:	PMC2685522
DOI/URL:	http://www.scopus.com/inward/record.url?eid=2-s2.0-66449107800&partnerID=40&md5=10df069ec1a7f0a2686460cc8714ec6a
Notes:	--- - "Cited By (since 1996): 1" - "Export Date: 30 November 2010" - "CODEN: RNARF" - "Source: Scopus"

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