The 1.15Å Crystal Structure of the Staphylococcus aureus Methionyl-aminopeptidase and Complexes with Triazole Based Inhibitors

Oefner, Christian; Douangamath, A.; D’Arcy, Allan; Häfeli, Sascha; Mareque, Daniel; Sweeney, A. Mac; Padilla, Juan; Pierau, Sabine; Schulz, Henk; Thormann, Michael; Wadman, Sjoerd; Dale, Glenn E.

doi:10.1016/s0022-2836(03)00862-3

Cited by 62 publications

(65 citation statements)

References 49 publications

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“…Undoubtedly, Co(II) is an excellent activator of purified MetAP apoenzyme, and the Co(II)-form MetAP is responsible for the discovery of most of the current MetAP inhibitors (22,23,(42)(43)(44) and has provided structural and mechanistic insight of the catalysis and inhibition of this therapeutically important family of enzymes (14). However, most Co(II) proteins are vitamin B12-dependent enzymes, and MetAP is among the limited number of enzymes that have Co(II) in a non-B12 form (45).…”

Section: Discussionmentioning

confidence: 99%

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FE(II) Is the Native Cofactor for Escherichia coli Methionine Aminopeptidase

Chai

Wang

2008

Journal of Biological Chemistry

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Section: Discussionmentioning

confidence: 99%

“…Potent MetAP inhibitors have been identified, but none of the inhibitors have shown significant antibacterial activity (13,22,23). Although most of the current MetAP inhibitors inhibit the Co(II)-form of MetAP effectively, their potency on other metalloforms often has not been characterized.…”

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confidence: 99%

FE(II) Is the Native Cofactor for Escherichia coli Methionine Aminopeptidase

Chai

Wang

2008

Journal of Biological Chemistry

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“…Therefore, human MetAPs may also serve as targets for the development of new anticancer agents. Some small molecules that inhibit MetAPs potently in vitro are known, but they lack potent antibacterial (10)(11)(12) or antiangiogenic (13) activities. One reason for this failure may be that they do not penetrate the bacterial or mammalian cells to reach their intended target, or perhaps they are efficiently transported back out of the cells.…”

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confidence: 99%

Structural basis of catalysis by monometalated methionine aminopeptidase

Xie

Qiang

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Methionine aminopeptidase (MetAP) removes the amino-terminal methionine residue from newly synthesized proteins, and it is a target for the development of antibacterial and anticancer agents. Available x-ray structures of MetAP, as well as other metalloaminopeptidases, show an active site containing two adjacent divalent metal ions bridged by a water molecule or hydroxide ion. The predominance of dimetalated structures leads naturally to proposed mechanisms of catalysis involving both metal ions. However, kinetic studies indicate that in many cases, only a single metal ion is required for full activity. By limiting the amount of metal ion present during crystal growth, we have now obtained a crystal structure for a complex of Escherichia coli MetAP with norleucine phosphonate, a transition-state analog, and only a single Mn(II) ion bound at the active site in the position designated M1, and three related structures of the same complex that show the transition from the mono-Mn(II) form to the di-Mn(II) form. An unliganded structure was also solved. In view of the full kinetic competence of the monometalated MetAP, the much weaker binding constant for occupancy of the M2 site compared with the M1 site, and the newly determined structures, we propose a revised mechanism of peptide bond hydrolysis by E. coli MetAP. We also suggest that the crystallization of dimetalated forms of metallohydrolases may, in some cases, be a misleading experimental artifact, and caution must be taken when structures are generated to aid in elucidation of reaction mechanisms or to support structure-aided drug design efforts.metalloprotease ͉ protein structure ͉ enzyme inhibition ͉ drug discovery ͉ metal occupancy A ll newly synthesized proteins have an amino-terminal methionine residue corresponding to the start codon AUG. In a significant number of cases, this initiating methionine residue is removed, either co-or posttranslationally, by the enzyme methionine aminopeptidase (MetAP) (1). In bacteria, this enzyme is the product of a single gene, and it is absolutely essential for bacterial survival, as demonstrated by gene deletion experiments in Escherichia coli (2) and Salmonella typhimurium (3). The single but critically essential MetAP enzyme of bacteria thus stands out as an attractive target for the design of antibacterial agents (4). Eukaryotes, on the other hand, have two distinct MetAPs, types I and II, arising from different genes (5). The human type II MetAP is a target of the antiangiogenic compounds fumagillin, ovalicin, and TNP-470 (6-8). Bengamides inhibit both types of MetAP (9) and cause inhibition of the growth of several human tumor cell lines in vitro at low-nanomolar concentrations. Therefore, human MetAPs may also serve as targets for the development of new anticancer agents. Some small molecules that inhibit MetAPs potently in vitro are known, but they lack potent antibacterial (10-12) or antiangiogenic (13) activities. One reason for this failure may be that they do not penetrate the bacterial or mammalian cells to...

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“…Prokaryotes have only one MetAP, either type I (eubacteria) or type II (archaea), whereas eukaryotic cells have both type I and type II MetAPs. Structural information is available for four MetAPs, namely the type I MetAPs from Escherichia coli (EcMetAP1) (7,8) and Staphylococcus aureus (9), and the type II MetAPs from Pyrococcus furiosis (10) and human (11). Locations of the S1 and S1Ј substrate binding pockets and the residues involved in forming these pockets have been assigned according to the structures of MetAPs complexed with the ligands derived from peptide substrates.…”

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Mutations at the S1 Sites of Methionine Aminopeptidases from Escherichia coli and Homo sapiens Reveal the Residues Critical for Substrate Specificity

Cui

Chen

et al. 2004

Journal of Biological Chemistry

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Methionine aminopeptidase (MetAP) catalyzes the removal of methionine from newly synthesized polypeptides. MetAP carries out this cleavage with high precision, and Met is the only natural amino acid residue at the N terminus that is accepted, although type I and type II MetAPs use two different sets of residues to form the hydrophobic S1 site. Characteristics of the S1 binding pocket in type I MetAP were investigated by systematic mutation of each of the seven S1 residues in Escherichia coli MetAP type I (EcMetAP1) and human MetAP type I (HsMetAP1). We found that Tyr-65 and Trp-221 in EcMetAP1, as well as the corresponding residues Phe-197 and Trp-352 in HsMetAP1, were essential for the hydrolysis of a thiopeptolide substrate, Met-S-Gly-Phe. Mutation of Phe-191 to Ala in HsMetAP1 caused inactivity in contrast to the full activity of EcMetAP1(Y62A), which may suggest a subtle difference between the two type I enzymes. The more striking finding is that mutation of Cys-70 in EcMetAP1 or Cys-202 in HsMetAP1 opens up the S1 pocket. The thiopeptolides Leu-S-GlyPhe and Phe-S-Gly-Phe, with previously unacceptable Leu or Phe as the N-terminal residue, became efficient substrates of EcMetAP1(C70A) and HsMetAP1(C202A). The relaxed specificity shown in these S1 site mutants for the N-terminal residues was confirmed by hydrolysis of peptide substrates and inhibition by reaction products. The structural features at the enzyme active site will be useful information for designing specific MetAP inhibitors for therapeutic applications.Removal of the N-terminal methionine from newly synthesized polypeptides is an important posttranslational modification and is catalyzed by methionine aminopeptidase (MetAP).

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The 1.15Å Crystal Structure of the Staphylococcus aureus Methionyl-aminopeptidase and Complexes with Triazole Based Inhibitors

Cited by 62 publications

References 49 publications

FE(II) Is the Native Cofactor for Escherichia coli Methionine Aminopeptidase

FE(II) Is the Native Cofactor for Escherichia coli Methionine Aminopeptidase

Structural basis of catalysis by monometalated methionine aminopeptidase

Mutations at the S1 Sites of Methionine Aminopeptidases from Escherichia coli and Homo sapiens Reveal the Residues Critical for Substrate Specificity

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