PDBsum entry: 1wkd

tRNA-modifying enzyme

PDB id: 1wkd

Contents

Protein chain

372 a.a. *

Metals

_ZN

Waters ×164

* Residue conservation analysis

PDB id:

1wkd

Name:

tRNA-modifying enzyme

Title:

tRNA-guanine transglycosylase

Structure:

tRNA-guanine transglycosylase. Chain: a. Synonym: tgt. Engineered: yes. Mutation: yes

Source:

Zymomonas mobilis. Organism_taxid: 542. Gene: tgt. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

2.60Å R-factor: 0.204 R-free: 0.276

Authors:

C.Romier,K.Reuter,D.Suck,R.Ficner

Key ref:

C.Romier et al. (1996). Mutagenesis and crystallographic studies of Zymomonas mobilis tRNA-guanine transglycosylase reveal aspartate 102 as the active site nucleophile. Biochemistry, 35, 15734-15739. PubMed id: 8961936 DOI: 10.1021/bi962003n

Date:

06-Aug-96 Release date: 07-Jul-97

Protein chain

P28720  (TGT_ZYMMO) -  Queuine tRNA-ribosyltransferase

Seq: 386 a.a.

Struc: 372 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.2.4.2.29 - tRNA-guanine transglycosylase.
• Reaction:
  1. [tRNA]-guanine + queuine = [tRNA]-queuine + guanine
  2. [tRNA]-guanine + 7-aminomethyl-7-carbaguanine = [tRNA]-7-aminomethyl- 7-carbaguanine + guanine

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 Gene Ontology (GO) functional annotation 

• Biological process: tRNA processing (3 terms)
• Biochemical function: transferase activity (4 terms)

reference

DOI no: 10.1021/bi962003n

Biochemistry 35:15734-15739 (1996)

PubMed id: 8961936

Mutagenesis and crystallographic studies of Zymomonas mobilis tRNA-guanine transglycosylase reveal aspartate 102 as the active site nucleophile.

C.Romier, K.Reuter, D.Suck, R.Ficner.

ABSTRACT

Procaryotic tRNA-guanine transglycosylase (TGT) catalyzes the posttranscriptional base exchange of the queuine precursor 7-aminomethyl-7-deazaguanine (preQ1) with the genetically encoded guanine at the wobble position of tRNAs specific for Asn, Asp, His, and Tyr. The X-ray structures of Zymomonas mobilis TGT and of its complex with preQ1 [Romier, C., Reuter, K., Suck, D., & Ficner, R. (1996) EMBO J. 15, 2850-2857] have revealed a specific preQ1 binding pocket and allowed a proposal for tRNA binding and recognition. We have used band-shift experiments in denaturing conditions to study the enzymatic reaction performed by TGT. The presence of shifted protein bands after incubation with tRNA followed by protein denaturation indicates a reaction mechanism involving a covalent intermediate. Inspection of the X-ray structures and comparison of the different procaryotic TGT sequences highlighted the conserved aspartate 102 as the most likely nucleophile. Mutation of this residue into alanine by site-directed mutagenesis leads to an inactive mutant unable to form a covalent intermediate with tRNA, proving that aspartate 102 is the active site nucleophile in TGT. To investigate the recognition of the wobble guanine in the preQ1 binding pocket, we mutated aspartate 156, the major recognition element for preQ1, into alanine and tyrosine. Both mutants are inactive in producing the final product, but the mutant D156A is able to form the covalent intermediate with tRNA in the first step of the reaction mechanism in comparable amounts to wild-type protein. Therefore, the binding of the wobble guanine in the preQ1 binding pocket is required for the cleavage of the glycosidic bond. The three mutants were crystallized and their X-ray structures determined. The mutants display only subtle changes to the wild-type protein, confirming that the observed biochemical results are due to the chemical substitutions rather than structural rearrangements.