PDBsum entry 1mc8

Hydrolase

PDB id: 1mc8

Contents

Protein chains

331 a.a. *

* Residue conservation analysis

PDB id:

1mc8

Name:

Hydrolase

Title:

Crystal structure of flap endonuclease-1 r42e mutant from pyrococcus horikoshii

Structure:

Flap endonuclease-1. Chain: a, b. Synonym: 5' nuclease. Engineered: yes. Mutation: yes

Source:

Pyrococcus horikoshii. Organism_taxid: 53953. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

3.10Å R-factor: 0.190 R-free: 0.279

Authors:

E.Matsui,K.V.Musti,J.Abe,K.Yamazaki,I.Matsui,K.Harata

Key ref:

E.Matsui et al. (2002). Molecular structure and novel DNA binding sites located in loops of flap endonuclease-1 from Pyrococcus horikoshii. J Biol Chem, 277, 37840-37847. PubMed id: 12147694 DOI: 10.1074/jbc.M205235200

Date:

06-Aug-02 Release date: 16-Oct-02

Protein chains

O50123  (FEN_PYRHO) -  Flap endonuclease 1 from Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)

Seq: 343 a.a.

Struc: 331 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.3.1.-.-

DOI no: 10.1074/jbc.M205235200

J Biol Chem 277:37840-37847 (2002)

PubMed id: 12147694

Molecular structure and novel DNA binding sites located in loops of flap endonuclease-1 from Pyrococcus horikoshii.

E.Matsui, K.V.Musti, J.Abe, K.Yamasaki, I.Matsui, K.Harata.

ABSTRACT

The crystal structure of flap endonuclease-1 from Pyrococcus horikoshii (phFEN-1) was determined to a resolution of 3.1 A. The active cleft of the phFEN-1 molecule is formed with one large loop and four small loops. We examined the function of the conserved residues and positively charged clusters on these loops by kinetic analysis with 45 different mutants. Arg(40) and Arg(42) on small loop 1, a cluster Lys(193)-Lys(195) on small loop 2, and two sites, Arg(94) and Arg(118)-Lys(119), on the large loop were identified as binding sites. Lys(87) on the large loop may play significant roles in catalytic reaction. Furthermore, we successfully elucidated the function of the four DNA binding sites that form productive ES complexes specific for each endo- or exo-type hydrolysis, probably by bending the substrates. For the endo-activity, Arg(94) and Lys(193)-Lys(195) located at the top and bottom of the molecule were key determinants. For the exo-activity, all four sites were needed, but Arg(118)-Lys(119) was dominant. The major binding sites for both the nick substrate and double-stranded DNA might be the same.

Selected figure(s)

Figure 1.
Fig. 1. The steric structure of a dimer phFEN-1 molecule. A, stereo view of the dimer. Red coils show helices (A-J) and blue arrows show strands (A-E). The figure was produced using the program MolScript. B, stereo view of the superimposition of P. horikoshii (red), P. furiosus (green), and M. jannaschii (blue) FEN-1s. The red and black arrows indicate the large loop and small loop 2, respectively. The figure was produced using the program Turbo-Frodo. C, stereo view of the dimer interface. The main chains from different subunits are distinguished with red and blue colors.

Figure 2.
Fig. 2. The location of the mutation on the structure. The number indicates the position of the mutation. Small loops 1 (39-55) and 2 (187-206), and the large loop (80-128) are colored yellow, green, and pink, respectively.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2002, 277, 37840-37847) copyright 2002.

Figures were selected by an automated process.