Functional categorization of the conserved basic amino acid residues in TrmH (tRNA (Gm18) methyltansferase) enzymes

Kazunori Watanabe, Osamu Nureki, Shuya Fukai, Yaeta Endo, Hiroyuki Hori

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Transfer RNA (Gm18) methyltransferase (TrmH) catalyzes the methyl transfer from S-adenosyl-L-methionine (AdoMet) to the 2′-OH group of the G18 ribose in tRNA. To identify amino acid residues responsible for the tRNA recognition, we have carried out the alanine substitution mutagenesis of the basic amino acid residues that are conserved only in TrmH enzymes and not in the other SpoU proteins. We analyzed the mutant proteins by S-adenosyl-L-homocysteine affinity column chromatography, gel mobility shift assay, and kinetic assay of the methyl transfer reaction. Based on these biochemical studies and the crystal structure of TrmH, we found that the conserved residues can be categorized according to their role (i) in the catalytic center (Arg-41), (ii) in the initial site of tRNA binding (Lys-90, Arg-166, Arg-168, and Arg-176), (iii) in the tRNA binding site required for continuation the catalytic cycle (Arg-8, Arg-19, and Lys-32), (iv) in the structural element involved in release of S-adenosyl-L-homocysteine (Arg-11-His-71-Met-147 interaction), (v) in the assisted phosphate binding site (His-34), or (vi) in an unknown function (Arg-109). Furthermore, the difference between the Kd and Km values for tRNA suggests that the affinity for tRNA is enhanced in the presence of AdoMet. To confirm this idea, we carried out the kinetic studies, a gel mobility shift assay with a mutant protein disrupted in the catalytic center, and the analytical gel-filtration chromatography. Our experimental results clearly show that the enzyme has a semi-ordered sequential mechanism in which AdoMet both enhances the affinity for tRNA and induces formation of the tetramer structure.

Original languageEnglish
Pages (from-to)34630-34639
Number of pages10
JournalJournal of Biological Chemistry
Volume281
Issue number45
DOIs
Publication statusPublished - Nov 10 2006
Externally publishedYes

Fingerprint

Basic Amino Acids
Transfer RNA
Enzymes
Binding Sites
Assays
Electrophoretic Mobility Shift Assay
Homocysteine
Mutant Proteins
Gels
Methionine
tRNA Methyltransferases
RNA, Transfer, Lys
S-Adenosylmethionine
Ribose
Affinity chromatography
Protein S
Mutagenesis
Column chromatography
Kinetics
Affinity Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Functional categorization of the conserved basic amino acid residues in TrmH (tRNA (Gm18) methyltansferase) enzymes. / Watanabe, Kazunori; Nureki, Osamu; Fukai, Shuya; Endo, Yaeta; Hori, Hiroyuki.

In: Journal of Biological Chemistry, Vol. 281, No. 45, 10.11.2006, p. 34630-34639.

Research output: Contribution to journalArticle

Watanabe, Kazunori ; Nureki, Osamu ; Fukai, Shuya ; Endo, Yaeta ; Hori, Hiroyuki. / Functional categorization of the conserved basic amino acid residues in TrmH (tRNA (Gm18) methyltansferase) enzymes. In: Journal of Biological Chemistry. 2006 ; Vol. 281, No. 45. pp. 34630-34639.
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AB - Transfer RNA (Gm18) methyltransferase (TrmH) catalyzes the methyl transfer from S-adenosyl-L-methionine (AdoMet) to the 2′-OH group of the G18 ribose in tRNA. To identify amino acid residues responsible for the tRNA recognition, we have carried out the alanine substitution mutagenesis of the basic amino acid residues that are conserved only in TrmH enzymes and not in the other SpoU proteins. We analyzed the mutant proteins by S-adenosyl-L-homocysteine affinity column chromatography, gel mobility shift assay, and kinetic assay of the methyl transfer reaction. Based on these biochemical studies and the crystal structure of TrmH, we found that the conserved residues can be categorized according to their role (i) in the catalytic center (Arg-41), (ii) in the initial site of tRNA binding (Lys-90, Arg-166, Arg-168, and Arg-176), (iii) in the tRNA binding site required for continuation the catalytic cycle (Arg-8, Arg-19, and Lys-32), (iv) in the structural element involved in release of S-adenosyl-L-homocysteine (Arg-11-His-71-Met-147 interaction), (v) in the assisted phosphate binding site (His-34), or (vi) in an unknown function (Arg-109). Furthermore, the difference between the Kd and Km values for tRNA suggests that the affinity for tRNA is enhanced in the presence of AdoMet. To confirm this idea, we carried out the kinetic studies, a gel mobility shift assay with a mutant protein disrupted in the catalytic center, and the analytical gel-filtration chromatography. Our experimental results clearly show that the enzyme has a semi-ordered sequential mechanism in which AdoMet both enhances the affinity for tRNA and induces formation of the tetramer structure.

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