Mechanism of Dihydrouridine Synthase 2 from Yeast and the Importance of Modifications for Efficient tRNA Reduction

Rider, Lance W.; Ottosen, M.; Gattis, Samuel G.; Palfey, Bruce A.

doi:10.1074/jbc.m806137200

Cited by 46 publications

(60 citation statements)

References 22 publications

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“…We recently found that TthDus expressed in E. coli forms a stable complex with tRNA in cells that cannot be dissociated even on SDS-PAGE (12). Rider, et al also reported similar results (11). In the present study, TthDus and Tth-tRNA Phe were coexpressed in E. coli and their complex was purified for structure analysis.…”

Section: Resultssupporting

confidence: 81%

“…Moreover, G19-C56 base pairing is prone to disruption in the transcripts as compared to modified tRNAs (18). Several previous studies indicated that Dus targets tRNA containing modifications (11,16). In this study, we showed that Dus can bind with tRNA that already has modifications other than D, but cannot recognize transcribed tRNA despite having the identical sequence (Fig.…”

Section: Resultsmentioning

confidence: 50%

“…The crystal structure of Dus from Thermotoga maritima has been reported (9), and mutation analysis of Dus from E. coli revealed important residues for dihydrouridine formation (10). One of the most remarkable biochemical features of Dus is that other modifications of tRNA are required for the enzymatic activity (11). However, the details of the reaction, including the tRNA recognition mechanism and its catalysis of dihydrouridine formation, are still unknown.…”

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confidence: 99%

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Molecular basis of dihydrouridine formation on tRNA

Tanaka

Yamashita

et al. 2011

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

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Dihydrouridine (D) is a highly conserved modified base found in tRNAs from all domains of life. Dihydrouridine synthase (Dus) catalyzes the D formation of tRNA through reduction of uracil base with flavin mononucleotide (FMN) as a cofactor. Here, we report the crystal structures of Thermus thermophilus Dus (TthDus), which is responsible for D formation at positions 20 and 20a, in complex with tRNA and with a short fragment of tRNA (D-loop). Dus interacts extensively with the D-arm and recognizes the elbow region composed of the kissing loop interaction between T-and D-loops in tRNA, pulling U20 into the catalytic center for reduction. Although distortion of the D-loop structure was observed upon binding of Dus to tRNA, the canonical D-loop/T-loop interaction was maintained. These results were consistent with the observation that Dus preferentially recognizes modified rather than unmodified tRNAs, indicating that Dus introduces D20 by monitoring the complete L-shaped structure of tRNAs. In the active site, U20 is stacked on the isoalloxazine ring of FMN, and C5 of the U20 uracil ring is covalently cross linked to the thiol group of Cys93, implying a catalytic mechanism of D20 formation. In addition, the involvement of a cofactor molecule in uracil ring recognition was proposed. Based on a series of mutation analyses, we propose a molecular basis of tRNA recognition and D formation catalyzed by Dus.protein-tRNA complex | RNA modification | substrate recognition | X-ray crystallography

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Section: Resultssupporting

confidence: 81%

Section: Resultsmentioning

confidence: 50%

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confidence: 99%

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Molecular basis of dihydrouridine formation on tRNA

Tanaka

Yamashita

et al. 2011

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

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“…S1A). The formation of dihydrouridine is catalyzed by dihydrouridine synthases (Dus) (10)(11)(12). Different Dus subfamilies display specificity toward distinct subsets of target uridines in tRNA.…”

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confidence: 99%

Major reorientation of tRNA substrates defines specificity of dihydrouridine synthases

Byrne

Jenkins

Peters

et al. 2015

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

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The reduction of specific uridines to dihydrouridine is one of the most common modifications in tRNA. Increased levels of the dihydrouridine modification are associated with cancer. Dihydrouridine synthases (Dus) from different subfamilies selectively reduce distinct uridines, located at spatially unique positions of folded tRNA, into dihydrouridine. Because the catalytic center of all Dus enzymes is conserved, it is unclear how the same protein fold can be reprogrammed to ensure that nucleotides exposed at spatially distinct faces of tRNA can be accommodated in the same active site. We show that the Escherichia coli DusC is specific toward U16 of tRNA. Unexpectedly, crystal structures of DusC complexes with tRNA Phe and tRNATrp show that Dus subfamilies that selectively modify U16 or U20 in tRNA adopt identical folds but bind their respective tRNA substrates in an almost reverse orientation that differs by a 160°rotation. The tRNA docking orientation appears to be guided by subfamily-specific clusters of amino acids ("binding signatures") together with differences in the shape of the positively charged tRNA-binding surfaces. tRNA orientations are further constrained by positional differences between the C-terminal "recognition" domains. The exquisite substrate specificity of Dus enzymes is therefore controlled by a relatively simple mechanism involving major reorientation of the whole tRNA molecule. Such reprogramming of the enzymatic specificity appears to be a unique evolutionary solution for altering tRNA recognition by the same protein fold.dihydrouridine synthase | tRNA modification | protein-RNA interaction | substrate specificity | X-ray crystallography D uring the posttranscriptional maturation of tRNA, about 10% of its nucleosides are enzymatically modified at specific positions (1). Altered levels of tRNA modification have been linked to several disorders including cancers (2-8). One of the most common modified nucleosides, dihydrouridine, is produced by reduction of the C5-C6 double bond in uridine. The resulting nonplanar base cannot form stabilizing stacking interactions with neighboring nucleotides and favors the C2′-endo ribose conformation (9). In Escherichia coli, dihydrouridine is commonly found at positions 16, 17, 20, and 20a of the D loop of tRNA (Fig. S1A). The formation of dihydrouridine is catalyzed by dihydrouridine synthases (Dus) (10-12). Different Dus subfamilies display specificity toward distinct subsets of target uridines in tRNA. Whereas the specificity of the four Saccharomyces cerevisiae Dus enzymes has been established (13), little is known about the three E. coli Dus proteins (DusA, DusB, and DusC), except that the specificities are nonoverlapping and that DusA modifies U20 (10). The mechanistic basis of the exquisite substrate specificity of Dus is an intriguing problem because the target uridines are exposed at spatially distinct faces of folded tRNAs, and yet all Dus subfamilies are predicted to adopt the same fold with highly conserved active-site residues (14-16).A stru...

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“…[4 S - 2 H]-NADPH was synthesized from [1- 2 H]glucose/hexokinase/glucose-6-phosphate dehydrogenase (13). …”

Section: Methodsmentioning

confidence: 99%

Mechanism of Flavin Reduction and Oxidation in the Redox-Sensing Quinone Reductase Lot6p fromSaccharomyces cerevisiae

et al. 2009

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Quinone reductases are flavin-containing enzymes that have been implicated in protecting organisms from redox stress and, more recently, as redox switches controlling the action of the proteasome. The reactions of the catalytic cycle of the dimeric quinone reductase Lot6p from Saccharomyces cerevisiae were studied in anaerobic stopped-flow experiments at 4° C. Both NADH and NADPH reacted similarly, reducing the FMN prosthetic group rapidly at saturation, but binding with very low affinity. The enzyme stereospecifically transferred the proS-hydride of NADPH with an isotope effect of 3.6, indicating that hydride transfer, and not an enzyme conformational change, is rate-determining in the reductive half-reaction. No intermediates such as charge-transfer complexes were detected. In the oxidative half-reaction, reduced enzyme reacted in a single phase with the six quinone substrates tested. The observed rate constants increased linearly with quinone concentration up to the limits allowed by solubility, indicating either a bimolecular reaction or very weak binding. The logarithm of the bimolecular rate constant increase linearly with the reduction potential of the quinone, consistent with the notion that quinone reductases strongly disfavor radical intermediates. Interestingly, both half-reactions of the catalytic cycle strongly resemble bioorganic model reactions; the reduction of Lot6p by NAD(P)H is moderately faster than non-enzymatic models, while the oxidation of Lot6p by quinones is actually slower than non-enzymatic reactions. This curious situation is consistent with the structure of Lot6p, which has a crease we propose to be the binding site for pyridine nucleotides, and space – but no obvious catalytic residues – near the flavin allowing the quinone to react. The decidedly sub-optimized catalytic cycle suggests that selective pressures other than maximizing quinone consumption shaped the evolution of Lot6p. This may reflect the importance of suppressing other potentially deleterious side-reactions, such as oxygen reduction, or it may indicate that the role Lot6p plays as a redox sensor in controlling the proteasome is more important than its role as a detoxifying enzyme.

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Mechanism of Dihydrouridine Synthase 2 from Yeast and the Importance of Modifications for Efficient tRNA Reduction

Cited by 46 publications

References 22 publications

Molecular basis of dihydrouridine formation on tRNA

Molecular basis of dihydrouridine formation on tRNA

Major reorientation of tRNA substrates defines specificity of dihydrouridine synthases

Mechanism of Flavin Reduction and Oxidation in the Redox-Sensing Quinone Reductase Lot6p fromSaccharomyces cerevisiae

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