PDBsum entry 1uq5

Hydrolase

PDB id: 1uq5

Contents

Protein chain

263 a.a. *

Ligands

ACT

SO4 ×2

Waters ×506

* Residue conservation analysis

PDB id:

1uq5

Name:

Hydrolase

Title:

Ricin a-chain (recombinant) n122a mutant

Structure:

Ricin. Chain: a. Fragment: a chain, residues 40-302. Engineered: yes. Mutation: yes

Source:

Ricinus communis. Castor bean. Organism_taxid: 3988. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PDB file)

Resolution:

1.40Å R-factor: 0.179 R-free: 0.214

Authors:

C.J.Marsden,V.Fulop

Key ref:

C.J.Marsden et al. (2004). The effect of mutations surrounding and within the active site on the catalytic activity of ricin A chain. Eur J Biochem, 271, 153-162. PubMed id: 14686928 DOI: 10.1046/j.1432-1033.2003.03914.x

Date:

15-Oct-03 Release date: 02-Jan-04

Protein chain

P02879  (RICI_RICCO) -  Ricin from Ricinus communis

Seq: 576 a.a.

Struc: 263 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.2.2.22 - rRNA N-glycosylase.
• Reaction: Endohydrolysis of the N-glycosidic bond at one specific adenosine on the 28S rRNA.

DOI no: 10.1046/j.1432-1033.2003.03914.x

Eur J Biochem 271:153-162 (2004)

PubMed id: 14686928

The effect of mutations surrounding and within the active site on the catalytic activity of ricin A chain.

C.J.Marsden, V.Fülöp, P.J.Day, J.M.Lord.

ABSTRACT

Models for the binding of the sarcin-ricin loop (SRL) of 28S ribosomal RNA to ricin A chain (RTA) suggest that several surface exposed arginine residues surrounding the active site cleft make important interactions with the RNA substrate. The data presented in this study suggest differing roles for these arginyl residues. Substitution of Arg48 or Arg213 with Ala lowered the activity of RTA 10-fold. Furthermore, substitution of Arg213 with Asp lowered the activity of RTA 100-fold. The crystal structure of this RTA variant showed it to have an unaltered tertiary structure, suggesting that the positively charged state of Arg213 is crucial for activity. Substitution of Arg258 with Ala had no effect on activity, although substitution with Asp lowered activity 10-fold. Substitution of Arg134 prevented expression of folded protein, suggesting a structural role for this residue. Several models have been proposed for the binding of the SRL to the active site of RTA in which the principal difference lies in the conformation of the second 'G' in the target GAGA motif in the 28S rRNA substrate. In one model, the sidechain of Asn122 is proposed to make interactions with this G, whereas another model proposes interactions with Asp75 and Asn78. Site-directed mutagenesis of these residues of RTA favours the first of these models, as substitution of Asn78 with Ser yielded an RTA variant whose activity was essentially wild-type, whereas substitution of Asn122 reduced activity 37.5-fold. Substitution of Asp75 failed to yield significant folded protein, suggesting a structural role for this residue.

Selected figure(s)

Figure 1.
Fig. 1. Models of hexanucleotide binding in the active site of RTA(based upon [10]). The structures are shown as stereo images with the C[1]G[2]A[3]G[4]A[5]G[6] (where A[3] is the target for depurination by RTA) in red. C[1] of the hexanucleotide is at the bottom left and the target adenine is at the top of each model. The sidechains of RTA are shown in blue. (A) Model 1 has the tetraloop bound in the active site of RTA with G[4] stacked upon the G[2]-A[5] pair and able to make interactions with Asn122. (B) Model 2 is a variation of model 1 with the tetraloop bound in a conformation where G[4] stacks with Tyr80 and makes interactions with Asp75 and Asn78. Drawn with MOLSCRIPT[34,35].

Figure 4.
Fig. 4. Electron density of RTA R213D in the vicinity of residue 213. The backbone and sidechains of the R213D substitution are shown as stereo images in thick ball and stick and the position of the Arg213 side-chain of the wild-type enzyme is overlayed and shown in thin ball and stick. The SIGMAA [33] weighted 2mF[o]- F[c] electron density using phases from the final model is contoured at 1 level, where represents the rms electron density for the unit cell. Contours more than 1.4 Å from any of the displayed atoms have been removed for clarity. Drawn with MOLSCRIPT[34,35].

The above figures are reprinted by permission from the Federation of European Biochemical Societies: Eur J Biochem (2004, 271, 153-162) copyright 2004.

Figures were selected by an automated process.