PDBsum entry 1b2m

Hydrolase/RNA

PDB id: 1b2m

Contents

Protein chains

104 a.a. *

Ligands

_DG-U34 ×3

Waters ×92

* Residue conservation analysis

PDB id:

1b2m

Name:

Hydrolase/RNA

Title:

Three-dimensional structure of ribonulcease t1 complexed with an isosteric phosphonate analogue of gpu: alternate substrate binding modes and catalysis.

Structure:

5'-r( Gp (U34))-3'. Chain: c, d, e. Engineered: yes. Ribonuclease t1. Chain: a, b. Mutation: yes. Other_details: rnase t1 complexed with 5'-r( Gp (Ch2)u)-3

Source:

Synthetic: yes. Aspergillus oryzae. Organism_taxid: 5062

Resolution:

2.00Å R-factor: 0.187 R-free: 0.254

Authors:

R.K.Arni,L.Watanabe,R.J.Ward,R.J.Kreitman,K.Kumar,F.G.Walz Jr.

Key ref:

R.K.Arni et al. (1999). Three-dimensional structure of ribonuclease T1 complexed with an isosteric phosphonate substrate analogue of GpU: alternate substrate binding modes and catalysis. Biochemistry, 38, 2452-2461. PubMed id: 10029539 DOI: 10.1021/bi982612q

Date:

27-Nov-98 Release date: 25-Mar-99

Protein chains

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

Seq: 130 a.a.

Struc: 104 a.a.

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]

DOI no: 10.1021/bi982612q

Biochemistry 38:2452-2461 (1999)

PubMed id: 10029539

Three-dimensional structure of ribonuclease T1 complexed with an isosteric phosphonate substrate analogue of GpU: alternate substrate binding modes and catalysis.

R.K.Arni, L.Watanabe, R.J.Ward, R.J.Kreitman, K.Kumar, F.G.Walz.

ABSTRACT

The X-ray crystal structure of a complex between ribonuclease T1 and guanylyl(3'-6')-6'-deoxyhomouridine (GpcU) has been determined at 2. 0 A resolution. This ligand is an isosteric analogue of the minimal RNA substrate, guanylyl(3'-5')uridine (GpU), where a methylene is substituted for the uridine 5'-oxygen atom. Two protein molecules are part of the asymmetric unit and both have a GpcU bound at the active site in the same manner. The protein-protein interface reveals an extended aromatic stack involving both guanines and three enzyme phenolic groups. A third GpcU has its guanine moiety stacked on His92 at the active site on enzyme molecule A and interacts with GpcU on molecule B in a neighboring unit via hydrogen bonding between uridine ribose 2'- and 3'-OH groups. None of the uridine moieties of the three GpcU molecules in the asymmetric unit interacts directly with the protein. GpcU-active-site interactions involve extensive hydrogen bonding of the guanine moiety at the primary recognition site and of the guanosine 2'-hydroxyl group with His40 and Glu58. On the other hand, the phosphonate group is weakly bound only by a single hydrogen bond with Tyr38, unlike ligand phosphate groups of other substrate analogues and 3'-GMP, which hydrogen-bonded with three additional active-site residues. Hydrogen bonding of the guanylyl 2'-OH group and the phosphonate moiety is essentially the same as that recently observed for a novel structure of a RNase T1-3'-GMP complex obtained immediately after in situ hydrolysis of exo-(Sp)-guanosine 2',3'-cyclophosphorothioate [Zegers et al. (1998) Nature Struct. Biol. 5, 280-283]. It is likely that GpcU at the active site represents a nonproductive binding mode for GpU [Steyaert, J., and Engleborghs (1995) Eur. J. Biochem. 233, 140-144]. The results suggest that the active site of ribonuclease T1 is adapted for optimal tight binding of both the guanylyl 2'-OH and phosphate groups (of GpU) only in the transition state for catalytic transesterification, which is stabilized by adjacent binding of the leaving nucleoside (U) group.