Hydrolysis of a slow cyclic thiophosphate substrate of RNase T1 analyzed by time-resolved crystallograph

Zegers, Ingrid; Loris, Remy; Dehollander, Gillis; Haikal, Abdelfattah; Poortmans, F.; Steyaert, Jan; Wyns, Lode

doi:10.1038/nsb0498-280

Cited by 36 publications

(31 citation statements)

References 28 publications

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“…Despite this, the structure of the active site remains very similar to that of the monomeric barnase. The exo-guanosine 2Ј,3Ј-phosphorothioate added in the crystallization setups was, unlike the situation with RNase T1 (19), not bound to the active site of barnase. Instead a sulfate anion makes contacts similar to those of the active site phosphate in the barnase⅐d(CGAC) complex (11).…”

Section: Resultsmentioning

confidence: 99%

Trimeric domain-swapped barnase

Zegers

Deswarte

Wyns

1999

Proc. Natl. Acad. Sci. U.S.A.

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The structure of a trimeric domain-swapped form of barnase (EC 3.1.27.3) was determined by x-ray crystallography at a resolution of 2.2 Å from crystals of space group R32. Residues 1-36 of one molecule associate with residues 41-110 from another molecule related through threefold symmetry. The resulting cyclic trimer contains three protein folds that are very similar to those in monomeric barnase. Both swapped domains contain a nucleation site for folding. The formation of a domain-swapped trimer is consistent with the description of the folding process of monomeric barnase as the formation and subsequent association of two foldons.

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Section: Resultsmentioning

confidence: 99%

Trimeric domain-swapped barnase

Zegers

Deswarte

Wyns

1999

Proc. Natl. Acad. Sci. U.S.A.

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“…For example, the colicin E5 active site includes a single arginine that coordinates both nonbridging oxygens of the scissile phosphodiester (Yajima et al 2006). The active site of RNase T1 also has a single arginine that contacts the scissile phosphodiester (Zegers et al 1998). By contrast, the active site of barnase includes two arginines (and a lysine) that contact the scissile phosphodiester (Buckle and Fersht 1994).…”

Section: Essential Argininesmentioning

confidence: 99%

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

Keppetipola

Jain²,

Meineke³

et al. 2009

RNA

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tRNA anticodon damage inflicted by secreted ribotoxins such as Kluyveromyces lactis g-toxin and bacterial colicins underlies a rudimentary innate immune system that distinguishes self from nonself species. The intracellular expression of g-toxin (a 232-amino acid polypeptide) arrests the growth of Saccharomyces cerevisiae by incising a single RNA phosphodiester 39 of the modified wobble base of tRNA Glu . Fungal g-toxin bears no primary structure similarity to any known nuclease and has no plausible homologs in the protein database. To gain insight to g-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 g-toxin). Structure-activity relations were gleaned from the effects of 44 conservative substitutions. Recombinant tag-free g-toxin, a monomeric protein, incised an oligonucleotide corresponding to the anticodon stem-loop of tRNA Glu at a single phosphodiester 39 of the wobble uridine. The anticodon nuclease was metal independent. RNA cleavage was abolished by ribose 29-H and 29-F modifications of the wobble uridine. Mutating His209 to alanine, glutamine, or asparagine abolished nuclease activity. We propose that g-toxin catalyzes an RNase A-like transesterification reaction that relies on His209 and a second nonhistidine side chain as general acid-base catalysts.

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“…1.5 mM protein and 3 mM substrate were incubated together for an hour at 20°C prior to adding an equal volume of mother liquor containing 2 mM CaCl 2 , 50 mM sodium acetate, pH 4.2, and 42-52% 2-methyl-2,4-pentanediol. Data collection and refinement procedures were carried out essentially as published (18); the results are summarized in Table I. The atomic coordinates for the H40A, E58A, and corresponding double mutant of RNase T 1 have been submitted to the Protein Data Bank, with respective entries 1LOW, 1LOV, 1LOY.…”

Section: Methodsmentioning

confidence: 99%

A Nucleophile Activation Dyad in Ribonucleases

Mignon¹,

Steyaert²,

Loris³

et al. 2002

Journal of Biological Chemistry

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Ribonucleases (RNases) catalyze the cleavage of the phosphodiester bond in RNA up to 10 15 -fold, as compared with the uncatalyzed reaction. High resolution crystal structures of these enzymes in complex with 3-mononucleotide substrates demonstrate the accommodation of the nucleophilic 2-OH group in a binding pocket comprising the catalytic base (glutamate or histidine) and a charged hydrogen bond donor (lysine or histidine). Ab initio quantum chemical calculations performed on such Michaelis complexes of the mammalian RNase A (EC 3.1.27.5) and the microbial RNase T 1 (EC 3.1.27.3) show negative charge build up on the 2-oxygen upon substrate binding. The increased nucleophilicity results from stronger hydrogen bonding to the catalytic base, which is mediated by a hydrogen bond from the charged donor. This hitherto unrecognized catalytic dyad in ribonucleases constitutes a general mechanism for nucleophile activation in both enzymic and RNAcatalyzed phosphoryl transfer reactions.RNases have been the subject of landmark research in areas ranging from basic protein chemistry to enzymology to protein folding and crystallography. These enzymes catalyze the intramolecular nucleophilic displacement of the 5Ј-leaving group by the attacking 2Ј-hydroxyl group in RNA, forming a 2Ј,3Ј-cyclophosphate (see Fig. 1). The nucleophilic attack occurs inline, in a postulated trigonal bipyramidal transition state, implying a catalytic base and acid on either side of the scissile bond (1, 2). X-ray crystallographical and site-directed mutagenesis experiments have provided substantial insight in the structure-function relationship of two unrelated families of ribonucleases. RNase A, (bovine pancreatic ribonuclease A) (EC 3.1.27.5) (3) and RNase T 1 , (EC 3.1.27.3) (4, 5) (and references therein) are the best characterized members of the mammalian and the microbial enzymes, respectively. The mammalian enzymes depend on two histidines for acid/base catalysis, whereas a histidine/glutamic acid pair is found in the microbial enzymes (6).The active site of RNase T 1 is composed of the side chains of Tyr-38, His-40, Glu-58, Arg-77, His-92, and Phe-100 (see Fig. 2a). With the exception of Arg-77, all these amino acids have been shown to take part in catalysis (5). Both the His-40 and Glu-58 side chains are in the direct vicinity of the 2Ј-nucleophile. pH dependence studies have shown Glu-58 to be unprotonated and His-40 to be protonated at the onset of catalysis, proving that Glu-58 is the catalytic base accepting a proton from the 2Ј-nucleophile (7). The protonated His-92 located at the opposite side of the active site functions as the catalytic acid. Removal of the His-40 side chain leads to a 6500-fold decrease in the second-order rate constant k cat /K m suggesting an important catalytic role for this residue in the wild type enzyme. His-40 is believed to polarize the 2Ј-hydroxyl group and to properly orientate Glu-58 toward the substrate (8, 9). However, the actual role of this residue is not totally clarified at present. Despite the l...

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Hydrolysis of a slow cyclic thiophosphate substrate of RNase T1 analyzed by time-resolved crystallograph

Cited by 36 publications

References 28 publications

Trimeric domain-swapped barnase

Trimeric domain-swapped barnase

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

A Nucleophile Activation Dyad in Ribonucleases

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