PDBsum entry 6rnt

Hydrolase(endoribonuclease)

PDB id: 6rnt

Contents

Protein chain

104 a.a. *

Ligands

2AM

Metals

_CA

Waters ×104

* Residue conservation analysis

PDB id:

6rnt

Name:

Hydrolase(endoribonuclease)

Title:

Crystal structure of ribonuclease t1 complexed with adenosine 2'- monophosphate at 1.8-angstroms resolution

Structure:

Ribonuclease t1. Chain: a. Engineered: yes

Source:

Aspergillus oryzae. Organism_taxid: 5062

Resolution:

1.80Å R-factor: 0.149

Authors:

J.Ding,G.Koellner,H.-P.Grunert,W.Saenger

Key ref:

J.Ding et al. (1991). Crystal structure of ribonuclease T1 complexed with adenosine 2'-monophosphate at 1.8-A resolution. J Biol Chem, 266, 15128-15134. PubMed id: 1651320

Date:

20-Aug-91 Release date: 15-Jan-93

Protein chain

P00651  (RNT1_ASPOR) -  Guanyl-specific ribonuclease T1 from Aspergillus oryzae (strain ATCC 42149 / RIB 40)

Seq: 130 a.a.

Struc: 104 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.4.6.1.24 - ribonuclease T1.
• Reaction: [RNA] containing guanosine + H2O = an [RNA fragment]-3'-guanosine- 3'-phosphate + a 5'-hydroxy-ribonucleotide-3'-[RNA fragment]

J Biol Chem 266:15128-15134 (1991)

PubMed id: 1651320

Crystal structure of ribonuclease T1 complexed with adenosine 2'-monophosphate at 1.8-A resolution.

J.Ding, G.Koellner, H.P.Grunert, W.Saenger.

ABSTRACT

Ribonuclease T1 was purified from an Escherichia coli overproducing strain and co-crystallized with adenosine 2'-monophosphate (2'-AMP) by microdialysis against 50% (v/v) 2-methyl-2,4-pentanediol in 20 mM sodium acetate, 2 mM calcium acetate, pH 4.2. The crystals have orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 48.93(1), b = 46.57(4), c = 41.04(2) A; Z = 4 and V = 93520 A3. The crystal structure was determined on the basis of the isomorphous structure of uncomplexed RNase T1 (Martinez-Oyanedel et al. (1991) submitted for publication) and refined by least squares methods using stereochemical restraints. The refinement was based on Fhkl of 7,445 reflections with Fo greater than or equal to 1 sigma (Fo) in the resolution range of 10-1.8 A, and converged at a crystallographic R factor of 0.149. The phosphate group of 2'-AMP is tightly hydrogen-bonded to the side chains of the active site residues Tyr38, His40, Glu58, Arg77, and His92, comparable with vanadate binding in the respective complex (Kostrewa, D., Choe, H.-W., Heinemann, U., and Saenger, W. (1989) Biochemistry 28, 7592-7600) and different from the complex with guanosine 2'-monophosphate (Arni, R., Heinemann, U., Tokuoka, R., and Saenger, W. (1988) J. Biol. Chem. 263, 15358-15368) where the phosphate does not interact with Arg77 and His92. The adenosine moiety is not located in the guanosine recognition site but stacked on Gly74 carbonyl and His92 imidazole, which serve as a subsite, as shown previously (Lenz, A., Cordes, F., Heinemann, U., and Saenger, W. (1991) J. Biol. Chem. 266, 7661-7667); in addition, there are hydrogen bonds adenine N6H . . . O Gly74 (minor component of three-center hydrogen bond) and adenosine O5' . . . O delta Asn36. These binding interactions readily explain why RNase T1 has some affinity for 2'-AMP. The molecular structure of RNase T1 is only marginally affected by 2'-AMP binding. Its "empty" guanosine-binding site features a flipped Asn43-Asn44 peptide bond and the side chains of Tyr45, Glu46 adopt conformations typical for RNase T1 not involved in guanosine binding. The side chains of amino acids Leu26, Ser35, Asp49, Val78 are disordered. The disorder of Val78 is of interest since this amino acid is located in a hydrophobic cavity, and the disorder appears to be correlated with an "empty" guanosine-binding site. The two Asp15 carboxylate oxygens and six water molecules coordinate a Ca2+ ion 8-fold in the form of a square antiprism.