Dataset Information

Insights into Catalytic and tRNA Recognition Mechanism of the Dual-Specific tRNA Methyltransferase from Thermococcus kodakarensis.

ABSTRACT: The tRNA methyltransferase Trm10, conserved throughout Eukarya and Archaea, catalyzes N1-methylation of purine residues at position 9 using S-adenosyl methionine as the methyl donor. The Trm10 family exhibits diverse target nucleotide specificity, with some homologs that are obligate m¹G? or m¹A?-specific enzymes, while others are bifunctional enzymes catalyzing both m¹G? and m¹A?. This variability is particularly intriguing given different chemical properties of the target N1 atom of guanine and adenine. Here we performed an extensive kinetic and mutational analysis of the m¹G? and m¹A?-catalyzing Trm10 from Thermococcus kodakarensis to gain insight into the active site that facilitates this unique bifunctionality. These results suggest that the rate-determining step for catalysis likely involves a conformational change to correctly position the substrate tRNA in the active site. In this model, kinetic preferences for certain tRNA can be explained by variations in the overall stability of the folded substrate tRNA, consistent with tRNA-specific differences in metal ion dependence. Together, these results provide new insight into the substrate recognition, active site and catalytic mechanism of m¹G/m¹A catalyzing bifunctional enzymes.

SUBMITTER: Krishnamohan A

PROVIDER: S-EPMC6410153 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Insights into Catalytic and tRNA Recognition Mechanism of the Dual-Specific tRNA Methyltransferase from <i>Thermococcus kodakarensis</i>.

Krishnamohan Aiswarya A Dodbele Samantha S Jackman Jane E JE

Genes 20190130 2

The tRNA methyltransferase Trm10, conserved throughout Eukarya and Archaea, catalyzes N1-methylation of purine residues at position 9 using <i>S</i>-adenosyl methionine as the methyl donor. The Trm10 family exhibits diverse target nucleotide specificity, with some homologs that are obligate m¹G₉ or m¹A₉-specific enzymes, while others are bifunctional enzymes catalyzing both m¹G₉ and m¹A₉. This variability is particularly intriguing given different chemical properties of the target N1 atom of gua ...[more]

PMID: 30704107

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Project description:tRNA m2G10/m22G10 methyltransferase (archaeal Trm11) methylates the 2-amino group in guanosine at position 10 in tRNA and forms N2,N2-dimethylguanosine (m22G10) via N2-methylguanosine (m2G10). We determined the complete sequence of tRNATrp, one of the substrate tRNAs for archaeal Trm11 from Thermococcus kodakarensis, a hyperthermophilic archaeon. Liquid chromatography/mass spectrometry following enzymatic digestion of tRNATrp identified 15 types of modified nucleoside at 21 positions. Several modifications were found at novel positions in tRNA, including 2'-O-methylcytidine at position 6, 2-thiocytidine at position 17, 2'-O-methyluridine at position 20, 5,2'-O-dimethylcytidine at position 32, and 2'-O-methylguanosine at position 42. Furthermore, methylwyosine was found at position 37 in this tRNATrp, although 1-methylguanosine is generally found at this location in tRNATrp from other archaea. We constructed trm11 (Δtrm11) and some gene disruptant strains and compared their tRNATrp with that of the wild-type strain, which confirmed the absence of m22G10 and other corresponding modifications, respectively. The lack of 2-methylguanosine (m2G) at position 67 in the trm11 trm14 double disruptant strain suggested that this methylation is mediated by Trm14, which was previously identified as an m2G6 methyltransferase. The Δtrm11 strain grew poorly at 95°C, indicating that archaeal Trm11 is required for T. kodakarensis survival at high temperatures. The m22G10 modification might have effects on stabilization of tRNA and/or correct folding of tRNA at the high temperatures. Collectively, these results provide new clues to the function of modifications and the substrate specificities of modification enzymes in archaeal tRNA, enabling us to propose a strategy for tRNA stabilization of this archaeon at high temperatures.IMPORTANCEThermococcus kodakarensis is a hyperthermophilic archaeon that can grow at 60 to 100°C. The sequence of tRNATrp from this archaeon was determined by liquid chromatography/mass spectrometry. Fifteen types of modified nucleoside were observed at 21 positions, including 5 modifications at novel positions; in addition, methylwyosine at position 37 was newly observed in an archaeal tRNATrp The construction of trm11 (Δtrm11) and other gene disruptant strains confirmed the enzymes responsible for modifications in this tRNA. The lack of 2-methylguanosine (m2G) at position 67 in the trm11 trm14 double disruptant strain suggested that this position is methylated by Trm14, which was previously identified as an m2G6 methyltransferase. The Δtrm11 strain grew poorly at 95°C, indicating that archaeal Trm11 is required for T. kodakarensis survival at high temperatures.

Dataset Information

Insights into Catalytic and tRNA Recognition Mechanism of the Dual-Specific tRNA Methyltransferase from Thermococcus kodakarensis.

Publications

Insights into Catalytic and tRNA Recognition Mechanism of the Dual-Specific tRNA Methyltransferase from <i>Thermococcus kodakarensis</i>.

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