PDBsum entry 1xo1

Hydrolase

PDB id: 1xo1

Contents

Protein chains

245 a.a. *

Waters ×316

* Residue conservation analysis

PDB id:

1xo1

Name:

Hydrolase

Title:

T5 5'-exonuclease mutant k83a

Structure:

5'-exonuclease. Chain: a, b. Synonym: 5'-nuclease. Engineered: yes. Mutation: yes

Source:

Enterobacteria phage t5. Organism_taxid: 10726. Strain: m72. Gene: d15. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.50Å R-factor: 0.226 R-free: 0.309

Authors:

T.A.Ceska,D.Suck,J.R.Sayers

Key ref:

S.J.Garforth et al. (1999). Mutagenesis of conserved lysine residues in bacteriophage T5 5'-3' exonuclease suggests separate mechanisms of endo-and exonucleolytic cleavage. Proc Natl Acad Sci U S A, 96, 38-43. PubMed id: 9874768 DOI: 10.1073/pnas.96.1.38

Date:

19-Nov-98 Release date: 02-Apr-99

Protein chains

P06229  (EXO5_BPT5) -  Flap endonuclease from Escherichia phage T5

Seq: 291 a.a.

Struc: 245 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.3.1.11.3 - exodeoxyribonuclease (lambda-induced).
• Reaction: Degradation of double-stranded DNA. It acts progressively in a 5'- to 3'-direction, releasing nucleoside 5'-phosphates.

DOI no: 10.1073/pnas.96.1.38

Proc Natl Acad Sci U S A 96:38-43 (1999)

PubMed id: 9874768

Mutagenesis of conserved lysine residues in bacteriophage T5 5'-3' exonuclease suggests separate mechanisms of endo-and exonucleolytic cleavage.

S.J.Garforth, T.A.Ceska, D.Suck, J.R.Sayers.

ABSTRACT

Efficient cellular DNA replication requires the activity of a 5'-3' exonuclease. These enzymes are able to hydrolyze DNA.DNA and RNA.DNA substrates exonucleolytically, and they are structure-specific endonucleases. The 5'-3' exonucleases are conserved in organisms as diverse as bacteriophage and mammals. Crystal structures of three representative enzymes identify two divalent-metal-binding sites typically separated by 8-10 A. Site-directed mutagenesis was used to investigate the roles of three lysine residues (K83, K196, and K215) situated near two metal-binding sites in bacteriophage T5 5'-3' exonuclease. Neither K196 nor K215 was essential for either the exo- or the endonuclease activity, but mutation of these residues increased the dissociation constant for the substrate from 5 nM to 200 nM (K196A) and 50 nM (K215A). Biochemical analysis demonstrated that K83 is absolutely required for exonucleolytic activity on single-stranded DNA but is not required for endonucleolytic cleavage of flap structures. Structural analysis of this mutant by x-ray crystallography showed no significant perturbations around the metal-binding sites in the active site. The wild-type protein has different pH optima for endonuclease and exonuclease activities. Taken together, these results suggest that different mechanisms for endo- and exonucleolytic hydrolysis are used by this multifunctional enzyme.

Selected figure(s)

Figure 1.
Fig. 1. The product distribution of T5 exonuclease reaction varies with pH. (A) Activity was assayed on the pseudo-Y substrate, with 10 mM MgCl[2] cofactor. Lanes marked M show the result of reactions in mixtures that contained no enzyme, and lanes 1-6 contained 4 nM wild-type enzyme in buffers at pH 5.5, 6.0, 7.0, 8.0, 9.3, and 11.3, respectively. The sizes (nucleotides) of the products are as indicated. (B) Time courses of wild-type 5'-3' exonuclease at pH extremes. Wild-type exonuclease, at a concentration of 0.8 nM, was incubated with the labeled pseudo-Y substrate at 37°C for 2.5, 5, or 10 min at pH 5.5 (lanes 1-3) or pH 9.3 (lanes 4-6) in the presence of 10 mM MgCl[2]. The products of the reaction were separated on a 7 M urea/15% acrylamide gel. Untreated substrate, lane M, and substrates incubated at 37°C for 10 min at each pH in the absence of enzyme (lanes 7 and 8) are shown. (C) Phosphoimager data from A showing the percentage of product plotted against pH for both exonucleolytic ( ) and endonucleolytic ( ) cleavage. (D) Graphical representation of the time course at pH 5.5 and 9.3 from data obtained from B. Endonucleolytic product is shown by dark bars, exonucleolytic product, by light bars. Differences between C and D in the absolute levels of exo- and endonuclease activity reflect the concentration of the enzyme used. In C all of the original substrate has been degraded.

Figure 5.
Fig. 5. (A) Electron density map of the region around the mutated residue K83. The density for this residue is defined in a 2F[o] F[c] map contoured at 1 . The positions of the next few residues of the native molecule are colored in brown, and these residues are disordered. The next residue is G84; this position is expected to show flexibility. The helical arch does not show evidence of order in the electron density map. The first well defined residue in helix 5 is F104. The absence of density preceding this residue suggests that the helix is unwound and exists in an undefined conformation. (B) A stereo view of the metal-binding site MeI as determined in the native molecule. The K83 side chain from the native molecule is shown in black. The positions of the metal's ligands are largely unchanged compared with the native (data not shown). The only amino group in close proximity to MeI is that of K83 (distance between NH[2] and MeI = 4.2 Å). (C) A C trace of the native (red) and K83A mutant structures (yellow) superimposed. Disordered residues in the mutant include 35-41 and 84-103. The regions around the acidic residues composing the metal-binding site are very similar. (D) A space-filling model of the protein, showing the three residues mutated in this study in red (top to bottom: K83, K196, and K215) in relation to the proposed threading mechanism. DNA is shown as colored strands and the metal sites observed are shown as purple spheres.

Figures were selected by an automated process.