PDBsum entry 3mat

Hydrolase/hydrolase inhibitor

PDB id: 3mat

Contents

Protein chain

264 a.a. *

Ligands

AHH-ALA-LEU

Metals

_NA

_CO ×2

Waters ×91

* Residue conservation analysis

PDB id:

3mat

Name:

Hydrolase/hydrolase inhibitor

Title:

E.Coli methionine aminopeptidase transition-state inhibitor complex

Structure:

Methionine aminopeptidase. Chain: a. Engineered: yes. Mutation: yes. Bestatin-based inhibitor (3r)-amino-(2s)-hydroxyheptanoyl- l-ala-l-leu-l-val-l-phe-ome. Chain: i. Engineered: yes. Other_details: synthetic

Source:

Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_cell_line: bl21(de3). Synthetic: yes

Resolution:

2.00Å R-factor: 0.156

Authors:

W.T.Lowther,A.M.Orville,D.T.Madden,S.Lim,D.H.Rich,B.W.Matthews

Key ref:

W.T.Lowther et al. (1999). Escherichia coli methionine aminopeptidase: implications of crystallographic analyses of the native, mutant, and inhibited enzymes for the mechanism of catalysis. Biochemistry, 38, 7678-7688. PubMed id: 10387007 DOI: 10.1021/bi990684r

Date:

29-Mar-99 Release date: 18-Jun-99

Protein chain

P0AE18  (MAP1_ECOLI) -  Methionine aminopeptidase from Escherichia coli (strain K12)

Seq: 264 a.a.

Struc: 264 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.3.4.11.18 - methionyl aminopeptidase.
• Reaction: Release of N-terminal amino acids, preferentially methionine, from peptides and arylamides.
• Cofactor: Cobalt cation

DOI no: 10.1021/bi990684r

Biochemistry 38:7678-7688 (1999)

PubMed id: 10387007

Escherichia coli methionine aminopeptidase: implications of crystallographic analyses of the native, mutant, and inhibited enzymes for the mechanism of catalysis.

W.T.Lowther, A.M.Orville, D.T.Madden, S.Lim, D.H.Rich, B.W.Matthews.

ABSTRACT

By improving the expression and purification of Escherichia coli methionine aminopeptidase (eMetAP) and using slightly different crystallization conditions, the resolution of the parent structure was extended from 2.4 to 1.9 A resolution. This has permitted visualization of the coordination geometry and solvent structure of the active-site dinuclear metal center. One solvent molecule (likely a mu-hydroxide) bridges the trigonal bipyramidal (Co1) and octahedral (Co2) cobalt ions. A second solvent (possibly a hydroxide ion) is bound terminally to Co2. A monovalent cation binding site was also identified about 13 A away from the metal center at an interface between the two subdomains of the protein. The first structure of a substrate-like inhibitor, (3R)-amino-(2S)-hydroxyheptanoyl-L-Ala-L-Leu-L-Val-L-Phe-OMe, bound to a methionine aminopeptidase, has also been determined. This inhibitor coordinates the metal center through four interactions as follows: (i) ligation of the N-terminal (3R)-nitrogen to Co2, (ii, iii) bridging coordination of the (2S)-hydroxyl group, and (iv) terminal ligation to Co1 by the keto oxygen of the pseudo-peptide linkage. Inhibitor binding occurs with the displacement of two solvent ligands and the expansion of the coordination sphere of Co1. In addition to the tetradentate, bis-chelate metal coordination, the substrate analogue forms hydrogen bonds with His79 and His178, two conserved residues within the active site of all MetAPs. To evaluate their importance in catalysis His79 and His178 were replaced with alanine. Both substitutions, but especially that of His79, reduce activity. The structure of the His79Ala apoenzyme and the comparison of its electronic absorption spectra with other variants suggest that the loss in activity is not due to a conformational change or a defective metal center. Two different reaction mechanisms are proposed and are compared to those of related enzymes. These results also suggest that inhibitors analogous to that reported here may be useful in preventing angiogenesis in cancer and in the treatment of microbial and fungal infections.