Control of Catalytic Cycle by a Pair of Analogous tRNA Modification Enzymes

Abstract

Enzymes that use distinct active site structures to perform identical reactions are known as analogous enzymes. The isolation of analogous enzymes suggests the existence of multiple enzyme structural pathways that can catalyze the same chemical reaction. A fundamental question concerning analogous enzymes is whether their distinct active-site structures would confer the same or different kinetic constraints to the chemical reaction, particularly with respect to the control of enzyme turnover. Here we address this question with the analogous enzymes of bacterial TrmD and its eukaryotic and archaeal counterpart Trm5. While both TrmD and Trm5 catalyze methyl transfer to synthesize the m1G37 base at the 3' position adjacent to the tRNA anticodon, using S-adenosyl methionine (AdoMet) as the methyl donor, TrmD features a trefoil-knot active-site structure whereas Trm5 features the Rossmann fold. Pre-steady-state analysis revealed that product synthesis by TrmD proceeds linearly with time, whereas that by Trm5 exhibits a rapid burst followed by a slower and linear increase with time. The burst kinetics of Trm5 suggests that product release is the rate-limiting step of the catalytic cycle, consistent with the observation of higher enzyme affinities to the products of tRNA and AdoMet. In contrast, the lack of burst kinetics of TrmD suggests that its turnover is controlled by a step required for product synthesis. Although TrmD exists as a homodimer, it showed "half-of-the-sites" reactivity for tRNA binding and product synthesis. The kinetic differences between TrmD and Trm5 are parallel to those between the two classes of aminoacyl-tRNA synthetases, which use distinct active-site structures to catalyze tRNA aminoacylation. This parallel suggests that the findings have a fundamental importance for enzymes that catalyze both methyl and aminoacyl transfer to tRNA in the decoding process.

Conservation of structure and mechanism by Trm5 enzymes

“…Fitting the data of k obs as a function of enzyme concentration to a hyperbola (Supplemental Fig. S4B) revealed the dissociation constant K d (AdoMet) (1.1 ± 0.1 μM), which is closely similar to the value of MjTrm5 (Christian et al 2010b). Similarly, fitting the data of k obs as a function of enzyme concentration in excess of tRNA and with saturating AdoMet revealed K d (tRNA) of 1.8 ± 0.1 μM (Supplemental Fig.…”

“…The affinity-purified HsTrm5 was examined for its steadystate activity on the transcript of human tRNA Cys , similar to the previous analysis of MjTrm5 with a transcript of the archaeal tRNA Cys (Christian et al 2004(Christian et al , 2010bSakaguchi et al 2012). The transcript of human tRNA…”

“…Eukaryotes and archaea employ the Trm5 enzyme (Bjork et al 2001;Christian et al 2004), whereas bacteria use the TrmD enzyme (Bystrom and Bjork 1982a,b). While both enzymes use AdoMet as the methyl donor to convert G37-tRNA to m 1 G37-tRNA, they are fundamentally distinct in structure (Ahn et al 2003;Christian et al 2004;Goto-Ito et al 2008, 2009, in kinetics (Christian et al 2010b), and in substrate recognition (Christian and Hou 2007;Lahoud et al 2011;Sakaguchi et al 2012). The lack of similarity between TrmD and Trm5 has led to the suggestion that specific targeting of TrmD, without affecting the Homo sapiens Trm5 (HsTrm5), would be a highly attractive strategy in developing the next generation of antibiotics (White and Kell 2004).…”

“…While eukaryotic and archaeal Trm5 enzymes are grouped as one family, the correlation between the two divisions in sequence, structure, and catalytic mechanism is not known. At present, the archaeal MjTrm5 (Methanococcus jannaschii) is the best-characterized enzyme, having an extensive set of kinetic data (Christian et al 2010b) and well-defined crystal structures, including a binary structure with sinefungin (an inactive AdoMet analog) and a ternary structure with AdoMet and tRNA (Goto-Ito et al 2008, 2009). However, whether MjTrm5 is a valid model to study HsTrm5 or other eukaryotic enzymes remains an open question and cannot be predicted based on sequence alone.…”

A novelHSD17B10mutation impairing the activities of the mitochondrial RNase P complex causes X-linked intractable epilepsy and neurodevelopmental regression

“…[40][41][42][43][44][45][46][47] The transcript of human mttRNA Leu (UUR) was used as the substrate for the synthesis of m 1 G9. The radioactive methyl donor was purchased from Perkin-Elmer.…”