PDBsum entry 1e5o

Hydrolase/hydrolase inhibitor

PDB id: 1e5o

Contents

Protein chain

330 a.a. *

Ligands

3AI

Waters ×189

* Residue conservation analysis

PDB id:

1e5o

Name:

Hydrolase/hydrolase inhibitor

Title:

Endothiapepsin complex with inhibitor db2

Structure:

Endothiapepsin. Chain: e. Fragment: residue 90-419. Ec: 3.4.23.23

Source:

Endothia parasitica. Organism_taxid: 5116

Biol. unit:

Monomer (from PDB file)

Resolution:

2.05Å R-factor: 0.181 R-free: 0.237

Authors:

J.A.Read,J.B.Cooper,L.Toldo,D.Bailey

Key ref:

G.W.Harris et al. (1994). Structure of the catalytic core of the family F xylanase from Pseudomonas fluorescens and identification of the xylopentaose-binding sites. Structure, 2, 1107-1116. PubMed id: 7881909 DOI: 10.1016/S0969-2126(94)00112-X

Date:

28-Jul-00 Release date: 07-Sep-00

Protein chain

P11838  (CARP_CRYPA) -  Endothiapepsin from Cryphonectria parasitica

Seq: 419 a.a.

Struc: 330 a.a.

Enzyme reactions

• Enzyme class: E.C.3.4.23.22 - endothiapepsin.
• Reaction: Hydrolysis of proteins with broad specificity similar to that of pepsin A, preferring hydrophobic residues at P1 and P1', but does not cleave 14-Ala-|-Leu-15 in the B chain of insulin or Z-Glu-Tyr. Clots milk.

DOI no: 10.1016/S0969-2126(94)00112-X

Structure 2:1107-1116 (1994)

PubMed id: 7881909

Structure of the catalytic core of the family F xylanase from Pseudomonas fluorescens and identification of the xylopentaose-binding sites.

G.W.Harris, J.A.Jenkins, I.Connerton, N.Cummings, L.Lo Leggio, M.Scott, G.P.Hazlewood, J.I.Laurie, H.J.Gilbert, R.W.Pickersgill.

ABSTRACT

BACKGROUND: Sequence alignment suggests that xylanases evolved from two ancestral proteins and therefore can be grouped into two families, designated F and G. Family F enzymes show no sequence similarity with any known structure and their architecture is unknown. Studies of an inactive enzyme-substrate complex will help to elucidate the structural basis of binding and catalysis in the family F xylanases. RESULTS: We have therefore determined the crystal structure of the catalytic domain of a family F enzyme, Pseudomonas fluorescens subsp. cellulosa xylanase A, at 2.5 A resolution and a crystallographic R-factor of 0.20. The structure was solved using an engineered catalytic core in which the nucleophilic glutamate was replaced by a cysteine. As expected, this yielded both high-quality mercurial derivatives and an inactive enzyme which enabled the preparation of the inactive enzyme-substrate complex in the crystal. We show that family F xylanases are eight-fold alpha/beta-barrels (TIM barrels) with two active-site glutamates, one of which is the nucleophile and the other the acid-base. Xylopentaose binds to five subsites A-E with the cleaved bond between subsites D and E. Ca2+ binding, remote from the active-site glutamates, stabilizes the structure and may be involved in the binding of extended substrates. CONCLUSIONS: The architecture of P. fluorescens subsp. cellulosa has been determined crystallographically to be a commonly occurring enzyme fold, the eight-fold alpha/beta-barrel. Xylopentaose binds across the carboxy-terminal end of the alpha/beta-barrel in an active-site cleft which contains the two catalytic glutamates.

Selected figure(s)

Figure 3.
Figure 3. Reaction mechanism for a retaining endo-β-1,4-xylanase. R is a number of xylose residues, HA is the acid catalyst. The structures in brackets are possible intermediates and R[1] is hydrogen or a number of xylose residues. Intermediate (a) has the nucleophile stabilizing the oxo-carbonium ion, whereas in (b) the covalent intermediate is formed. Either of these intermediates could react with water and be hydrolyzed or react with another xylo-oligosaccharide to produce trans-glycosylation products. Figure 3. Reaction mechanism for a retaining endo-β-1,4-xylanase. R is a number of xylose residues, HA is the acid catalyst. The structures in brackets are possible intermediates and R[1] is hydrogen or a number of xylose residues. Intermediate (a) has the nucleophile stabilizing the oxo-carbonium ion, whereas in (b) the covalent intermediate is formed. Either of these intermediates could react with water and be hydrolyzed or react with another xylo-oligosaccharide to produce trans-glycosylation products.

Figure 9.
Figure 9. The averaged 3.0 Å electron-density map, in the region of β-strand 5 showing the quality of the map and the density for aromatics which were used to initially align the sequence with the electron-density map. The map is contoured at 1σ. Figure 9. The averaged 3.0 Å electron-density map, in the region of β-strand 5 showing the quality of the map and the density for aromatics which were used to initially align the sequence with the electron-density map. The map is contoured at 1σ.

The above figures are reprinted by permission from Cell Press: Structure (1994, 2, 1107-1116) copyright 1994.

Figures were selected by an automated process.