In Vitro Activity of the RrmJ Mutants

To analyze the influence of the amino acid substitutions on the methyltransfer activity of RrmJ, we first needed to characterize the enzymatic properties of wild type RrmJ (Hager et al., 2002). The apparent Vmax of the methyltransfer reaction, as well as the apparent Km values for 50S ribosomal subunits and AdoMet, were determined by in vitro methylation assays using purified RrmJ and radioactively labeled AdoMet. 50S ribosomal subunits were prepared from the rrmJ deletion strain HB23, because their 23S rRNA lacks the U₂₅₅₂ methylation catalyzed by RrmJ. The assay conditions have been established (see “Materials and Methods”) in which the rate of [³H]methyl incorporation from [³H]AdoMet into 23S rRNA was proportional to the RrmJ concentration and linear over more than 150 min (data not shown). Thus, the initial rate measurements represented true initial velocities, and the de-pendence of the rate on substrate concentration could be measured. The apparent Km value for 23S rRNA within 50S ribosomal subunits was determined to be 0.8 ⁺ 0.1 µM with an apparent Kcat of 0.064 min^-1 at 37ºC. The apparent Km for AdoMet was determined to be 3.7 ⁺ 0.3 µM (Hager et al., 2002).

To characterize the in vitro methylation activity of the mutant proteins, the specific activity of the purified RrmJ mutant proteins was determined under conditions in which the two substrates, 50S ribosomes and AdoMet, are present in saturating concentrations (see Fig.

10 and Table 1). Both lysine mutants, K38A and K164A, showed an at least a 50-fold decrease in their in vitro methylation activity. The very small amount of methyl incorporation observed was within the limits of the background incorporation, and an increase in enzyme concentration did not cause any increase in methyl incorporation (data not shown). This showed that these two lysine residues play a critical role in the methyltransfer reaction and corroborated the in vivo data that suggested that both residues are important for the overall function of RrmJ. Substitution of E199 caused a 16-fold reduction in Kcat without changing the Km for 50S ribosomal subunits. This suggested a less important role for this residue in the catalytic mechanism, an assumption that agreed well with the phenotype of rrmJ deletion strains expressing this E199A mutant protein. These cells showed only slightly affected polysome profiles and minor growth disadvantages on McConkey plates suggesting that the activity of this mutant protein is sufficient in vivo to partly rescue the rrmJ deletion.

Figure 10: Apparent Kcat values of the inactive mutants in RrmJ (Hager et al., 2002)

100 nM purified enzyme was incubated with 50 µM ³H-AdoMet (200 µC/ml) in methylation buffer (50 mM HEPES•KOH, pH 7.5, 100 mM NH4Cl, 3 mM MgCl2, 2 mM β-mercaptoethanol; 40 U RNasIn) at 37°C. The methylation reaction was started by the addition of 5 µM 50S ribosomal subunits prepared from the rrmJ deletion strain HB23. At defined time points, aliquots were taken and the ³H-methyl incorporation was determined as described.

To ascertain that the E199A mutant protein shows indeed sufficient in vivo methylation activity to account for the rescued phenotype, the extent of 23S rRNA modification in 50S ribosomal subunits prepared from this mutant strain was analyzed, by

testing them as substrates in the in vitro methylation assay (Fig. 11). If the E199A mutant was fully active in vivo, most of the 50S ribosomal subunits should be already methylated, and the in vitro methyl incorporation should be as low as the methyl incorporation observed with 50S ribosomal subunits prepared from the wild type strain HB24. If the E199A mutant was inactive in vivo, on the other hand, the in vitro methylation should be as substantial as the in vitro methylation of 50S ribosomal subunits prepared from the rrmJ deletion strain HB23. More than 60% of 50S ribosomal subunits prepared from HB23 strains expressing the E199A mutant were methylated and no longer served as in vitro substrate for RrmJ. These results confirmed the in vivo data that revealed the presence of a substantial amount of stable 50S ribosomal subunits and suggested a considerable methylation activity of the E199A mutant in vivo.

Figure 11: In vitro methylation of 50S prepared from the E199A mutant strain

300 nM purified enzyme was incubated with 50 µM ³H-AdoMet (200 µC/ml) in methylation buffer (50 mM HEPES•KOH, pH 7.5, 100 mM NH4Cl, 3 mM MgCl2, 2 mM β-mercaptoethanol;

40 U RNasIn) at 37°C. The methylation reaction was started by the addition of 2 µM 50S ribosomal subunits prepared from the rrmJ deletion strain HB23, the rrmJ wild type strain HB24 and the rrmJ deletion strain expressing the RrmJ-E199A mutant from a plasmid (JuH 26). After 14 hours the total ³ H-methyl incorporation was measured.

The specific activity of the Y201A mutant protein was not as severely impaired as the activity of the K38A and K138A mutant proteins, suggesting that this residue does not take a direct part in the methyltransfer reaction (Fig. 10). Surprisingly, the D83A mutant protein that showed no activity in vivo showed about 20% of wild type activity in vitro, which has been determined to be 0.064 min^-1. This may reflect the fact that this mutant shows an increase in Km for both substrates. While the apparent Kmvalues of wild type RrmJ for 50S ribosomal subunits and for AdoMet were determined to be 0.8 ⁺ 0.1 µM and 3.7 ⁺ 0.3 µM,

respectively (Hager et al., 2002), the D83A mutant revealed an apparent Km of 7.0 µM for 50S ribosomal subunits and an apparent Km of 1.4 µM for AdoMet, leading to a more significant decrease in catalytic activity at lower substrate concentrations, that might be present in the cell.

Table 1: Summary of the in vivo and in vitro characterization of RrmJ’s active site mutants (Hager et al., 2002)

Mutation Growth 37°C

Polysome profile

AdoMet bound [%]

app. Kcat

[min^-1]

app. Km (50S) [µM]

wild type +++ +++ 76 0.064 0.8

D83A - - 0 0.012 1.4^a

K38A - - 68 < 0.001 n.d.

D124A (+) (+) n.d.^b n.d.^b n.d.^b

K164A - - 74 < 0.001 n.d.

E199A ++ ++ 72 0.004 0.7

Y201A ++ ++ 66 0.012 0.5

a The apparent Km for AdoMet was determined to be 7.0 µM.

b D124A mutant aggregated upon overexpression in BL21rrmJ∆567cells and could not be purified.

This mutant protein was soluble in the strains that were used to investigate the in vivo activity of the protein.

The in vivo data presented for wild type RrmJ were obtained with the rrmJ deletion strain HB23 expressing wild type rrmJ from a pET 11a plasmid (HB25). Growth of this strain is very similar to that of the corresponding wild type E. coli strain (MG1655) but it accumulates slightly higher levels of 30S and 50S ribosomal subunits in lysates prepared under associating conditions and 40S ribosomal subunits in lysates prepared under dissociating salt conditions. Western blot analysis revealed that all mutant variants were soluble and expressed in similar amounts as the single copy of wild type RrmJ from its normal chromosomal location in MG1655. ND, not determined; -, (+), ++ or +++, ribosome profiles are severely, moderately, slightly, or not impaired in lysates prepared under the indicated salt conditions.