Insights into folate/FAD-dependent tRNA methyltransferase mechanism: role of two highly conserved cysteines in catalysis

Abstract

The flavoprotein TrmFO methylates specifically the C5 carbon of the highly conserved uridine 54 in tRNAs. Contrary to most methyltransferases, the 1-carbon unit transferred by TrmFO derives from 5,10-methylenetetrahydrofolate and not from S-adenosyl-L-methionine. The enzyme also employs the FAD hydroquinone as a reducing agent of the C5 methylene U54-tRNA intermediate in vitro. By analogy with the catalytic mechanism of thymidylate synthase ThyA, a conserved cysteine located near the FAD isoalloxazine ring was proposed to act as a nucleophile during catalysis. Here, we mutated this residue (Cys-53 in Bacillus subtilis TrmFO) to alanine and investigated its functional role. Biophysical characterization of this variant demonstrated the major structural role of Cys-53 in maintaining both the integrity and plasticity of the flavin binding site. Unexpectedly, gel mobility shift assays showed that, like the wild-type enzyme, the inactive C53A variant was capable of forming a covalent complex with a 5-fluorouridine-containing mini-RNA. This result confirms the existence of a covalent intermediate during catalysis but rules out a nucleophilic role for Cys-53. To identify the actual nucleophile, two other strictly conserved cysteines (Cys-192 and Cys-226) that are relatively far from the active site were replaced with alanine, and a double mutant C53A/C226A was generated. Interestingly, only mutations that target Cys-226 impeded TrmFO from forming a covalent complex and methylating tRNA. Altogether, we propose a revised mechanism for the m(5)U54 modification catalyzed by TrmFO, where Cys-226 attacks the C6 atom of the uridine, and Cys-53 plays the role of the general base abstracting the C5 proton.

SCHEME 1.

A, proposed mechanism for the reaction catalyzed by TrmFO. R, p-aminobenzoylglutamate. Note that some steps of this mechanism are similar to those of thymidylate synthase ThyA. The reaction starts with the reduction of FAD to FADH₂ by NAD(P)H, followed by the dissociation of NAD(P)⁺ and subsequent binding of the methylene donor CH₂THF. All of the following steps of the mechanism are detailed in the Introduction. B, proposed formation of a ternary covalent complex between TrmFO, CH₂THF, and a C5-fluorinated uridine-containing mini-RNA substrate analog.

FIGURE 1.

Structure of TrmFO_TT (Protein Data Bank code 3G5U) showing the two-domain architecture of the protein. The flavin domain is shown in green, and the insertion domain is shown in magenta. The FAD cofactor is in yellow, and the two tryptophan residues, Trp-18 and Trp-283, are in green and magenta, respectively. The details of the flavin binding site showing the residues conserved across the TrmFO family are depicted in the lower panel. The three conserved cysteines, including Cys-51, which is located less than 3 Å away from the isoalloxazine ring, are shown as sticks.

FIGURE 2.

tRNA binding and activity of wild-type and C53A TrmFO_BS. A, comparison of the tRNA methylation activity of wild-type and C53A TrmFO_BS. E. coli [α-³²P]UTP-labeled tRNA^Asp transcript was incubated at 37 °C with wild-type or C53A TrmFO_BS at 37 °C in 50 mm HEPES-Na, pH 7.5, 100 mm ammonium sulfate, 0.1 mm EDTA, 25 mm mercaptoethanol, 0.5 mm NAD(P)H, 0.5 mm CH₂THF, and 20% glycerol. After incubation, the tRNA transcript was digested by nuclease P1, and the resulting nucleotides were analyzed by two-dimensional TLC on cellulose plates and autoradiography. B, determination of the dissociation constants of wild-type and C53A TrmFO_BS for E. coli tRNA^Asp using a nitrocellulose binding assay.

FIGURE 3.

Covalent catalysis by wild-type and C53A TrmFO_BS. A, sequence and secondary structures of 31-mer B. subtilis 5-FU-mini-RNA substrate analog. B, formation of a covalent complex between wild-type or C53A TrmFO_BS and 5-FU-mini-RNA substrate. The formation of the covalent complex was analyzed by 10% SDS-PAGE after Coomassie Blue staining. Lane 1, markers; lane 2, C53A TrmFO_BS alone; lane 3, C53A TrmFO_BS + 5-FU-mini-RNA; lane 4, C53A TrmFO_BS + CH₂THF + 5-FU-mini-RNA; lane 5, C53A TrmFO_BS + NADH + CH₂THF + 5-FU-mini-RNA; lane 6, wild-type TrmFO_BS alone; lane 7, wild-type TrmFO_BS + 5-FU-mini-RNA. C, purification of the covalent complex on a MonoQ anion exchange column. The absorbance was followed simultaneously at 260 nm (RNA; gray solid line), 280 nm (protein; dotted line), and 450 nm (FAD; black solid line). The linear NaCl gradient is shown in dashed lines.

FIGURE 4.

A, effect of iodoacetamide on covalent complex formation in C53A TrmFO_BS. Lane 1, C53A TrmFO_BS alone; lane 2, C53A TrmFO_BS + 5-FU-mini-RNA; lane 3, C53A TrmFO_BS + 5-FU-mini-RNA + 10 mm iodoacetamide; lane 4, C53A TrmFO_BS + 5-FU-mini-RNA + 50 mm iodoacetamide; lane 5, C53A TrmFO_BS + 5-FU-mini-RNA + 100 mm iodoacetamide. B, formation of the covalent complex between various TrmFO_BS mutants and 5-FU-mini-RNA. C, limited proteolysis of wild-type and C53A TrmFO_BS in the presence and absence of bulk tRNA. Digested protein (7.5 μg in each sample) was loaded on a 12% SDS-polyacrylamide gel and analyzed by Coomassie Blue staining. Lane 1, wild-type TrmFO_BS; lane 2, wild-type TrmFO_BS + trypsin with boxes indicating the band of full-length protein subjected to mass spectrometry analysis and that of the most abundant truncated form (ΔTrmFO_BS); lane 3, wild-type TrmFO_BS complexed to bulk tRNA + trypsin; 4, C53A TrmFO_BS; lane 5, C53A TrmFO_BS + trypsin; lane 6, C53A TrmFO_BS complexed to bulk tRNA + trypsin. Note that the incubation of TrmFO_BS with bulk tRNA in the absence of trypsin leads to a profile similar to that of TrmFO_BS alone. D, limited proteolysis of wild-type and C53A TrmFO_BS in the presence and absence of E. coli tRNA^Asp transcript. Lane 1, wild-type TrmFO_BS alone; lane 2, wild type TrmFO_BS + trypsin; lane 3, markers; lane 4, wild-type TrmFO_BS complexed to tRNA^Asp + trypsin; lane 5, C53A TrmFO_BS; lane 6, C53A TrmFO_BS + trypsin; lane 7, C53A TrmFO_BS complexed to tRNA^Asp + trypsin.

FIGURE 5.

MALDI peptide mass fingerprinting analysis of the most abundant truncated form of TrmFO_BS after mild trypsinolysis. A, protein coverage of TrmFO_BS is shown in boldface type with four peptides present in low abundance in the ΔTrmFO_BS spectrum underlined. Squared arginine indicates the cleavage site induced by mild trypsinolysis. B, mass spectrum profile of peptides obtained after complete digestion of the full-length (top) and truncated TrmFO_BS (bottom) by AspN protease.

FIGURE 6.

Flavin and tryptophan accessibilities determined using fluorescence quenching in wild-type and C53A TrmFO_BS. The quenching was performed in 100 mm sodium phosphate buffer, pH 8, by adding small aliquots of KI stock solution to 800 μl of sample containing ∼6 μm TrmFO_BS. A, Stern-Volmer plot of FAD fluorescence quenching by KI of wild-type TrmFO_BS (circles) and C53A mutant (squares) monitored at 529 nm. B, Stern-Volmer plot of tryptophan (Trp-20 and Trp-286) fluorescence quenching by KI of wild-type TrmFO_BS (circles) and C53A mutant (squares) monitored at 349 nm. The solid lines represent fits to Equation 2.

FIGURE 7.

A, normalized fluorescence emission transition curves for the pressure-induced unfolding of wild-type and C53A TrmFO_BS. B, pressure dependence of the free energy corresponding to the unfolding equilibrium of TrmFO_BS. Pressure is given in MPa units (1 bar = 0.1 MPa = 105 kg/ms², corresponding to the hybrid unit of 1.02 kg/cm²).