Specificity and kinetics of 23S rRNA modification enzymes RlmH and RluD

Abstract

Along the ribosome assembly pathway, various ribosomal RNA processing and modification reactions take place. Stem-loop 69 in the large subunit of Escherichia coli ribosomes plays a substantial role in ribosome functioning. It contains three highly conserved pseudouridines synthesized by pseudouridine synthase RluD. One of the pseudouridines is further methylated by RlmH. In this paper we show that RlmH has unique substrate specificity among rRNA modification enzymes. It preferentially methylates pseudouridine and less efficiently uridine. Furthermore, RlmH is the only known modification enzyme that is specific to 70S ribosomes. Kinetic parameters determined for RlmH are the following: The apparent K M for substrate 70S ribosomes is 0.51 6 0.06 mM, and for cofactor S-adenosyl-L-methionine 27 6 3 mM; the k cat values are 4.95 6 1.10 min À1 and 6.4 6 1.3 min À1, respectively. Knowledge of the substrate specificity and the kinetic parameters of RlmH made it possible to determine the kinetic parameters for RluD as well. The K M value for substrate 50S subunits is 0.98 6 0.18 mM and the k cat value is 1.97 6 0.46 min À1. RluD is the first rRNA pseudouridine synthase to be kinetically characterized. The determined rates of RluD-and RlmH-directed modifications of 23S rRNA are compatible with the rate of 50S assembly in vivo. The fact that RlmH requires 30S subunits demonstrates the dependence of 50S subunit maturation on the simultaneous presence of 30S subunits.

Small methyltransferase RlmH assembles a composite active site to methylate a ribosomal pseudouridine

“…These values, measured using N-terminal His-tagged RlmH, agree well with reported values for untagged RlmH 33 ( K M for 70S = 0.51 ± 0.06 µM, k cat  = 4.95 ± 1.10 min −1 ). The catalytic activity of RlmH in our study ( k cat / K M  ≥ 8.3 min −1  · µM −1 ) is therefore similar to that of native RlmH ( k cat / K M  = 9.71 min −1  · µM −1 ) 33 .

Figure 2Kinetic analyses of wild-type and mutant RlmH. Initial velocities ( V 0 , µM min −1 ) of methylation of Δ rlmH −70S ribosome (0.3 µM) by wild-type or mutant RlmH at 25 °C with 50 µM [ 3 H]-labeled SAM.

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“…We monitored methylation using 50 µM [ 3 H]-SAM. The SAM concentration was > K M for SAM 33 , consistent with the occupancy of one binding site per RlmH monomer. Testing whether occupancy of the second SAM binding site affects the rate of methylation was not feasible in the current assay.…”

Substrate Recognition and Modification by a Pathogen-Associated Aminoglycoside Resistance 16S rRNA Methyltransferase

“…S1 in the supplemental material) and analysis of multiple variants. While several characterized rRNA-modifying enzymes do appear to have substantially lower substrate affinities (26)(27)(28)(29), there is also precedence for very-high-affinity 16S rRNA methyltransferase-substrate binding in the case of RsmA (KsgA), which binds pre-30S as part of a subunit assembly quality control mechanism (30, 31). For NpmA, a high 30S affinity might be important for its ability to compete for its substrate with the other abundant binding partners in the bacterial cell, such as translation initiation factors or the 50S subunit.…”

Quantitative Analysis of rRNA Modifications Using Stable Isotope Labeling and Mass Spectrometry

“…11−13 Detailed investigations of RluD- and RlmH-dependent modifications in vivo and in vitro suggest that pseudouridylations of 1911, 1915, and 1917 occur at the stage of completion of the 50S assembly and Ψ(1915) is methylated during or after subunit joining. 12,13,40 As in case of the 16S residue m 4 Cm(1402), qMS may not resolve the RluD and RlmH steps, as the corresponding modifications are monitored using the same nucleolytic fragments (Table 2), requiring more in depth analyses.…”