PDBsum entry 1wkm

Hydrolase

PDB id: 1wkm

Contents

Protein chains

295 a.a. *

Ligands

MET ×2

Metals

_MN ×4

Waters ×325

* Residue conservation analysis

PDB id:

1wkm

Name:

Hydrolase

Title:

The product bound form of the mn(ii)loaded methionine aminopeptidase from hyperthermophile pyrococcus furiosus

Structure:

Methionine aminopeptidase. Chain: a, b. Synonym: map, peptidase m. Engineered: yes

Source:

Pyrococcus furiosus. Organism_taxid: 2261. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.30Å R-factor: 0.215 R-free: 0.252

Authors:

A.J.Copik,B.P.Nocek,S.B.Jang,S.I.Swierczek,S.Ruebush,L.Meng, V.M.D'Souza,J.W.Peters,B.Bennett,R.C.Holz

Key ref:

A.J.Copik et al. (2005). EPR and X-ray crystallographic characterization of the product-bound form of the MnII-loaded methionyl aminopeptidase from Pyrococcus furiosus. Biochemistry, 44, 121-129. PubMed id: 15628852 DOI: 10.1021/bi048123+

Date:

01-Jun-04 Release date: 22-Feb-05

Protein chains

P56218  (MAP2_PYRFU) -  Methionine aminopeptidase from Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)

Seq: 295 a.a.

Struc: 295 a.a.

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/bi048123+

Biochemistry 44:121-129 (2005)

PubMed id: 15628852

EPR and X-ray crystallographic characterization of the product-bound form of the MnII-loaded methionyl aminopeptidase from Pyrococcus furiosus.

A.J.Copik, B.P.Nocek, S.I.Swierczek, S.Ruebush, S.B.Jang, L.Meng, V.M.D'souza, J.W.Peters, B.Bennett, R.C.Holz.

ABSTRACT

Methionine aminopeptidases (MetAPs) are ubiquitous metallohydrolases that remove the N-terminal methionine from nascent polypeptide chains. Although various crystal structures of MetAP in the presence of inhibitors have been solved, the structural aspects of the product-bound step has received little attention. Both perpendicular- and parallel-mode electron paramagnetic resonance (EPR) spectra were recorded for the Mn(II)-loaded forms of the type-I (Escherichia coli) and type-II (Pyrococcus furiosus) MetAPs in the presence of the reaction product l-methionine (L-Met). In general, similar EPR features were observed for both [MnMn(EcMetAP-I)]-L-Met and [MnMn(PfMetAP-II)]-L-Met. The observed perpendicular-mode EPR spectra consisted of a six-line hyperfine pattern at g = 2.03 (A = 8.8 mT) with less intense signals with eleven-line splitting at g = 2.4 and 1.7 (A = 4.4 mT). The former feature results from mononuclear, magnetically isolated Mn(II) ions and this signal are 3-fold more intense in the [MnMn(PfMetAP-II)]-L-Met EPR spectrum than in the [MnMn(EcMetAP-I)]-L-Met spectrum. Inspection of the EPR spectra of both [MnMn(EcMetAP-I)]-L-Met and [MnMn(PfMetAP-II)]-L-Met at 40 K in the parallel mode reveals that the [Mn(EcMetAP-I)]-L-Met spectrum exhibits a well-resolved hyperfine split pattern at g = 7.6 with a hyperfine splitting constant of A = 4.4 mT. These data suggest the presence of a magnetically coupled dinuclear Mn(II) center. On the other hand, a similar feature was not observed for the [MnMn(PfMetAP-II)]-L-Met complex. Therefore, the EPR data suggest that L-Met binds to [MnMn(EcMetAP-I)] differently than [MnMn(PfMetAP-II)]. To confirm these data, the X-ray crystal structure of [MnMn(PfMetAP-II)]-L-Met was solved to 2.3 A resolution. Both Mn1 and Mn2 reside in a distorted trigonal bipyramidal geometry, but the bridging water molecule, observed in the [CoCo(PfMetAP-II)] structure, is absent. Therefore, L-Met binding displaces this water molecule, but the carboxylate oxygen atom of L-Met does not bridge between the two Mn(II) ions. Instead, a single carboxylate oxygen atom of L-Met interacts with only Mn1, while the N-terminal amine nitrogen atom binds to M2. This L-Met binding mode is different from that observed for L-Met binding [CoCo(EcMetAP-I)]. Therefore, the catalytic mechanisms of type-I MetAPs may differ somewhat from type-II enzymes when a dinuclear metalloactive site is present.