PDBsum entry 2iq6

Hydrolase

PDB id: 2iq6

Contents

Protein chain

291 a.a. *

Ligands

LEU-LEU-LEU

Metals

_ZN ×2

Waters ×259

* Residue conservation analysis

PDB id:

2iq6

Name:

Hydrolase

Title:

Crystal structure of the aminopeptidase from vibrio proteolyticus in complexation with leucyl-leucyl-leucine.

Structure:

Bacterial leucyl aminopeptidase. Chain: a. Peptide, (leucyl-leucyl-leucine). Chain: b. Engineered: yes

Source:

Vibrio proteolyticus. Organism_taxid: 671. Synthetic: yes. Synthetic construct. Organism_taxid: 32630. Other_details: the peptide was chemically synthesized.

Resolution:

2.00Å R-factor: 0.206 R-free: 0.247

Authors:

A.Kumar,B.Narayanan,J.-J.P.Kim,B.Bennett

Key ref:

A.Kumar et al. (2007). Experimental evidence for a metallohydrolase mechanism in which the nucleophile is not delivered by a metal ion: EPR spectrokinetic and structural studies of aminopeptidase from Vibrio proteolyticus. Biochem J, 403, 527-536. PubMed id: 17238863

Date:

13-Oct-06 Release date: 28-Aug-07

Protein chain

Q01693  (AMPX_VIBPR) -  Bacterial leucyl aminopeptidase from Vibrio proteolyticus

Seq: 504 a.a.

Struc: 291 a.a.

Enzyme reactions

• Enzyme class: E.C.3.4.11.10 - bacterial leucyl aminopeptidase.
• Reaction: Release of an N-terminal amino acid, preferentially leucine, but not glutamic or aspartic acids.
• Cofactor: Zn(2+)

Biochem J 403:527-536 (2007)

PubMed id: 17238863

Experimental evidence for a metallohydrolase mechanism in which the nucleophile is not delivered by a metal ion: EPR spectrokinetic and structural studies of aminopeptidase from Vibrio proteolyticus.

A.Kumar, G.R.Periyannan, B.Narayanan, A.W.Kittell, J.J.Kim, B.Bennett.

ABSTRACT

Metallohydrolases catalyse some of the most important reactions in biology and are targets for numerous chemotherapeutic agents designed to combat bacterial infectivity, antibiotic resistance, HIV infectivity, tumour growth, angiogenesis and immune disorders. Rational design of inhibitors of these enzymes with chemotherapeutic potential relies on detailed knowledge of the catalytic mechanism. The roles of the catalytic transition ions in these enzymes have long been assumed to include the activation and delivery of a nucleophilic hydroxy moiety. In the present study, catalytic intermediates in the hydrolysis of L-leucyl-L-leucyl-L-leucine by Vibrio proteolyticus aminopeptidase were characterized in spectrokinetic and structural studies. Rapid-freeze-quench EPR studies of reaction products of L-leucyl-L-leucyl-L-leucine and Co(II)-substituted aminopeptidase, and comparison of the EPR data with those from structurally characterized complexes of aminopeptidase with inhibitors, indicated the formation of a catalytically competent post-Michaelis pre-transition state intermediate with a structure analogous to that of the inhibited complex with bestatin. The X-ray crystal structure of an aminopeptidase-L-leucyl-L-leucyl-L-leucine complex was also analogous to that of the bestatin complex. In these structures, no water/hydroxy group was observed bound to the essential metal ion. However, a water/hydroxy group was clearly identified that was bound to the metal-ligating oxygen atom of Glu152. This water/hydroxy group is proposed as a candidate for the active nucleophile in a novel metallohydrolase mechanism that shares features of the catalytic mechanisms of aspartic proteases and of B2 metallo-beta-lactamases. Preliminary studies on site-directed variants are consistent with the proposal. Other features of the structure suggest roles for the dinuclear centre in geometrically and electrophilically activating the substrate.