PDBsum entry 1rn4

Hydrolase(endoribonuclease)

PDB id: 1rn4

Contents

Protein chain

100 a.a. *

Ligands

PO4

Waters ×82

* Residue conservation analysis

PDB id:

1rn4

Name:

Hydrolase(endoribonuclease)

Title:

His92ala mutation in ribonuclease t1 induces segmental flexibility. An x-ray study

Structure:

Ribonuclease t1. Chain: a. Engineered: yes

Source:

Aspergillus oryzae. Organism_taxid: 5062. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.80Å R-factor: 0.148

Authors:

W.Saenger,G.Koellner

Key ref:

G.Koellner et al. (1992). His92Ala mutation in ribonuclease T1 induces segmental flexibility. An X-ray study. J Mol Biol, 224, 701-713. PubMed id: 1314902 DOI: 10.1016/0022-2836(92)90554-W

Date:

07-Nov-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: 100 a.a.*

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

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: 10.1016/0022-2836(92)90554-W

J Mol Biol 224:701-713 (1992)

PubMed id: 1314902

His92Ala mutation in ribonuclease T1 induces segmental flexibility. An X-ray study.

G.Koellner, H.W.Choe, U.Heinemann, H.P.Grunert, A.Zouni, U.Hahn, W.Saenger.

ABSTRACT

In the genetically mutated ribonuclease T1 His92Ala (RNase T1 His92Ala), deletion of the active site His92 imidazole leads to an inactive enzyme. Attempts to crystallize RNase T1 His92Ala under conditions used for wild-type enzyme failed, and a modified protocol produced two crystal forms, one obtained with polyethylene glycol (PEG), and the other with phosphate as precipitants. Space groups are identical to wild-type RNase T1, P2(1)2(1)2(1), but unit cell dimensions differ significantly, associated with different molecular packings in the crystals; they are a = 31.04 A, b = 62.31 A, c = 43.70 A for PEG-derived crystals and a = 32.76 A, b = 55.13 A, c = 43.29 A for phosphate-derived crystals, compared to a = 48.73 A, b = 46.39 A, c = 41.10 A for uncomplexed wild-type RNase T1. The crystal structures were solved by molecular replacement and refined by stereochemically restrained least-squares methods based on Fo greater than or equal to sigma (Fo) of 3712 reflections in the resolution range 10 to 2.2 A (R = 15.8%) for the PEG-derived crystal and based on Fo greater than or equal to sigma (Fo) of 6258 reflections in the resolution range 10 to 1.8 A (R = 14.8%) for the phosphate-derived crystal. The His92Ala mutation deletes the hydrogen bond His92N epsilon H ... O Asn99 of wild-type RNase T1, thereby inducing structural flexibility and conformational changes in the loop 91 to 101 which is located at the periphery of the globular enzyme. This loop is stabilized in the wild-type protein by two beta-turns of which only one is retained in the crystals obtained with PEG. In the crystals grown with phosphate as precipitant, both beta-turns are deleted and the segment Gly94-Ala95-Ser96-Gly97 is so disordered that it is not seen at all. In addition, the geometry of the guanine binding site in both mutant studies is different from "empty" wild-type RNase T1 but similar to that found in complexes with guanosine derivatives: the Glu46 side-chain carboxylate hydrogen bonds to Tyr42 O eta; water molecules that are present in the guanine binding site of "empty" wild-type RNase T1 are displaced; the Asn43-Asn44 peptide is flipped such that phi/psi-angles of Asn44 are in alpha L-conformation (that is observed in wild-type enzyme when guanine is bound).(ABSTRACT TRUNCATED AT 400 WORDS)

Selected figure(s)

Figure 1.
Figure 1. Stereo drawings of the orientation of the RNase T, molecules in the unit cel (drawn with SCHAKAL: Keller, 1988). For clarity, only C'' atoms are shown; the loop 91 to 101 s drawn in thick lines. (a) RNase T, wild-type. Xote that the terminus Ala1 NH; of each molecule contacts the loop 91 to 101 of a symmetry-related molecule. (b) RNase T, H92A-PEG. With respect to the wild-type structure, the u-axis is shortened ad the h-axis is lengthened. Loop 91 to 101 contacts the a-helix of a symmetry-related molecule. (c) Rru'ase T, HOBA-phosphate. With respect to (b), the b-axis is shortened. Segment 94 to 97 o loop 91 to 101 was located due to disorder and is therefore not drawn.

Figure 4.
Figure 4. Electron density map (2F,- F,) of the dis- ordered disulfide bridge Cys2Sy-CyslOSy in RNase T, H92A-phosphate.

The above figures are reprinted by permission from Elsevier: J Mol Biol (1992, 224, 701-713) copyright 1992.

Figures were selected by an automated process.