A single amino acid difference between archaeal and human type 2 methionine aminopeptidases differentiates their affinity towards ovalicin

In almost all living cells, methionine aminopeptidase (MetAP) co-translationally cleaves the initiator methionine in at least 70% of the newly synthesized polypeptides. MetAPs are typically classified into Type 1 and Type 2. While prokaryotes and archaea contain only either Type 1 or Type 2 MetAPs respectively, eukaryotes contain both types of enzymes. Almost all MetAPs published till date cleave only methionine from the amino terminus of the substrate peptides. Earlier experiments on crude Type 2a MetAP isolated from Pyrococcus furiosus (PfuMetAP2a) cosmid protein library was shown to cleave leucine in addition to methionine. Authors in that study have ruled out the PfuMetAP2a activity against leucine substrates and assumed it to be a background reaction contributed by other contaminating proteases. In the current paper, using the pure recombinant enzyme, we report that indeed activity against leucine is directly carried out by the PfuMetAP2a. In addition, the natural product ovalicin which is a specific covalent inhibitor of Type 2 MetAPs does not show efficient inhibition against the PfuMetAP2a. Bioinformatic analysis suggested that a glycine in eukaryotic MetAP2s (G222 in human MetAP2b) and asparagine (N53 in PfuMetAP2a) in archaeal MetAP2s positioned at the analogous position. N53 side chain forms a hydrogen bond with a conserved histidine (H62) at the entrance of the active site and alters its orientation to accommodate the ovalicin. This slight orientational difference of the H62, reduces affinity of the ovalicin by 300,000-fold when compared with the HsMetAP2b inhibition. This difference in the activity is partly reduced in the case of N53G mutation of the PfuMetAP2a.     

Structure of the angiogenesis inhibitor ovalicin bound to its noncognate target, human Type 1 methionine aminopeptidase

Methionine aminopeptidases (MetAPs) remove the initiator methionine during protein biosynthesis. They exist in two isoforms, MetAP1 and MetAP2. The anti-angiogenic compound fumagillin binds tightly to the Type 2 MetAPs but only weakly to Type 1. High-affinity complexes of fumagillin and its relative ovalicin with Type 2 human MetAP have been reported. Here we describe the crystallographic structure of the low-affinity complex between ovalicin and Type 1 human MetAP at 1.1 A resolution. This provides the first opportunity to compare the structures of ovalicin or fumagillin bound to a Type 1 and a Type 2 MetAP. For both Type 1 and Type 2 human MetAPs the inhibitor makes a covalent adduct with a corresponding histidine. At the same time there are significant differences in the alignment of the inhibitors within the respective active sites. It has been argued that the lower affinity of ovalicin and fumagillin for the Type 1 MetAPs is due to the smaller size of their active sites relative to the Type 2 enzymes. Comparison with the uncomplexed structure of human Type 1 MetAP indicates that there is some truth to this. Several active site residues have to move "outward" by 0.5 Angstroms or so to accommodate the inhibitor. Other residues move "inward." There are, however, other factors that come into play. In particular, the side chain of His310 rotates by 134 degrees into a different position where (together with Glu128 and Tyr195) it coordinates a metal ion not seen at this site in the native enzyme.     

Expression and characterization of Mycobacterium tuberculosis methionine aminopeptidase type 1a

Methionine aminopeptidase (MetAP) carries out the cotranslational N-terminal methionine excision and is essential for bacterial survival. Mycobacterium tuberculosis expresses two MetAPs, MtMetAP1a and MtMetAP1c, at different levels in growing and stationary phases, and both are potential targets to develop novel antitubercular therapeutics. Recombinant MtMetAP1a was purified as an apoenzyme, and metal binding and activation were characterized with an activity assay using a fluorogenic substrate. Ni(II), Co(II) and Fe(II) bound tightly at micromolar concentrations, and Ni(II) was the most efficient activator for the MetAP-catalyzed substrate hydrolysis. Although the characteristics of metal binding and activation are similar to MtMetAP1c we characterized before, MtMetAP1a was significantly more active, and more importantly, a set of inhibitors displayed completely different inhibitory profiles on the two mycobacterial MetAPs in both potency and metalloform selectivity. The differences in catalysis and inhibition predicted the significant differences in active site structure.     

Two continuous spectrophotometric assays for methionine aminopeptidase

Two spectrophotometric assays have been developed for methionine aminopeptidases (MetAPs). The first method employs a thioester substrate which, upon enzymatic removal of the N-terminal methionine, generates a free thiol group. The released thiol is quantitated using Ellman's reagent. The MetAP reaction is conveniently monitored on a UV-VIS spectrophotometer in a continuous fashion, with the addition of an excess of Ellman's reagent into the assay reaction. Two tripeptide analogues were synthesized and found to be excellent substrates of both Escherichia coli MetAP and human MetAP2 (k(cat)/K(M) = 2.8 x 10(5) M(-1) s(-1) for the most reactive substrate). In the second assay method, the MetAP reaction is coupled to a prolyl aminopeptidase reaction using Met-Pro-p-nitroanilide as substrate. MetAP-catalyzed cleavage of the N-terminal methionine produces prolyl-p-nitroanilide, which is rapidly hydrolyzed by the prolyl aminopeptidase from Bacillus coagulans to release a chromogenic product, p-nitroaniline. This allows the MetAP reaction to be continuously monitored at 405 nm on a UV-VIS spectrophotometer. The assays have been applied to determine the pH optima and kinetic constants for the E. coli and human MetAPs as well as to screen MetAP inhibitors. These results demonstrate that the current assays are convenient, rapid, and sensitive methods for kinetic studies of MetAPs and effective tools for screening MetAP inhibitors.     

Pyridinylpyrimidines selectively inhibit human methionine aminopeptidase-1

Cellular protein synthesis is initiated with methionine in eukaryotes with few exceptions. Methionine aminopeptidases (MetAPs) which catalyze the process of N-terminal methionine excision are essential for all organisms. In mammals, type 2 MetAP (MetAP2) is known to be important for angiogenesis, while type 1 MetAP (MetAP1) has been shown to play a pivotal role in cell proliferation. Our previous high-throughput screening of a commercial compound library uncovered a novel class of inhibitors for both human MetAP1 (HsMetAP1) and human MetAP2 (HsMetAP2). This class of inhibitors contains a pyridinylpyrimidine core. To understand the structure–activity relationship (SAR) and to search for analogues of 2 with greater potency and higher HsMetAP1-selectivity, a total of 58 analogues were acquired through either commercial source or by in-house synthesis and their inhibitory activities against HsMetAP1 and HsMetAP2 were determined. Through this systematic medicinal chemistry analysis, we have identified (1) 5-chloro-6-methyl-2-pyridin-2-ylpyrimidine as the minimum element for the inhibition of HsMetAP1; (2) 5′-chloro as the favored substituent on the pyridine ring for the enhanced potency against HsMetAP1; and (3) long C4 side chains as the essentials for higher HsMetAP1-selectivity. At the end of our SAR campaign, 25b, 25c, 26d and 30a–30c are among the most selective and potent inhibitors of purified HsMetAP1 reported to date. In addition, we also performed crystallographic analysis of one representative inhibitor (26d) in complex with N-terminally truncated HsMetAP1.     

Identification of an SH3-binding motif in a new class of methionine aminopeptidases from Mycobacterium tuberculosis suggests a mode of interaction with the ribosome

The crystal structure of the methionine aminopeptidase (MetAP) from Mycobacterium tuberculosis (MtMetAP1c) has been determined in the apo- and methionine-bound forms. This is the first structure of a type I MetAP with a significant extension at the amino terminus. The catalytic domain is similar to that of Escherichia coli MetAP (EcMetAP), and the additional 40-residue segment wraps around the surface with an extended but well-defined structure. There are several members of the actinomyces family of bacteria that contain MetAPs with such N-terminal extensions, and we classify these as MetAP type Ic (MetAP1c). Some members of this family of bacteria also contain a second MetAP (type Ia) similar in size to EcMetAP. The main difference between the apo- and the methionine-bound forms of MtMetAP1c is in the conformation of the metal-binding residues. The position of the methionine bound in the active site is very similar to that found in many of the known members of this family. Side chains of several residues in the S1 and S1' subsites shift as much as 1.5 A compared to EcMetAP. Residues 14-17 have the sequence Pro-Thr-Arg-Pro and adopt the conformation of a polyproline II helix. Model-building suggests that this PxxP segment can bind to an SH3 protein motif. Other type Ib and type Ic MetAPs with N-terminal extensions contain similarly located PxxP motifs. Also, several ribosomal proteins are known to include SH3 domains, one of which is located close to the tunnel from which the nascent polypeptide chain exits the ribosome. Therefore, it is proposed that the binding of MetAPs to the ribosome is mediated by a complex between a PxxP motif on the protein and an SH3 domain on the ribosome. It is also possible that zinc-finger domains, which are located at the extreme N-terminus of type I MetAPs, may participate in interactions with the ribosome.     

Analyzing the binding of Co(II)-specific inhibitors to the methionyl aminopeptidases from Escherichia coli and Pyrococcus furiosus

Methionine aminopeptidases (MetAPs) represent a unique class of protease that is capable of the hydrolytic removal of an N-terminal methionine residue from nascent polypeptide chains. MetAPs are physiologically important enzymes; hence, there is considerable interest in developing inhibitors that can be used as antiangiogenic and antimicrobial agents. A detailed kinetic and spectroscopic study has been performed to probe the binding of a triazole-based inhibitor and a bestatin-based inhibitor to both Mn(II)- and Co(II)-loaded type-I (Escherichia coli) and type-II (Pyrococcus furiosus) MetAPs. Both inhibitors were found to be moderate competitive inhibitors. The triazole-type inhibitor was found to interact with both active-site metal ions, while the bestatin-type inhibitor was capable of switching its mode of binding depending on the metal in the active site and the type of MetAP enzyme.     

Structural basis for the functional differences between type I and type II human methionine aminopeptidases

Determination of the crystal structure of human MetAP1 makes it possible, for the first time, to compare the structures of a Type I and a Type II methionine aminopeptidase (MetAP) from the same organism. Comparison of the Type I enzyme with the previously reported complex of ovalicin with Type II MetAP shows that the active site of the former is reduced in size and would incur steric clashes with the bound inhibitor. This explains why ovalicin and related anti-angiogenesis inhibitors target Type II human MetAP but not Type I. The differences in both size and shape of the active sites between MetAP1 and MetAP2 also help to explain their different substrate specificity. In the presence of excess Co(2+), a third cobalt ion binds in the active site region, explaining why metal ions in excess can be inhibitory. Also, the N-terminal region of the protein contains three distinct Pro-x-x-Pro motifs, supporting the prior suggestion that this region of the protein may participate in binding to the ribosome.     

A new colorimetric assay for methionyl aminopeptidases: examination of the binding of a new class of pseudopeptide analog inhibitors

A direct and convenient spectrophotometric assay has been developed for methionine aminopeptidases (MetAPs). The method employs the hydrolysis of a substrate that is a methionyl analogue of p-nitroaniline (L-Met-p-NA), which releases the chromogenic product p-nitroaniline. This chromogenic product can be monitored continuously using a UV-Vis spectrophotometer set at 405 nm. The assay was tested with the type I MetAP from Escherichia coli (EcMetAP-I) and the type II MetAP from Pyrococcus furiosus (PfMetAP-II). Using L-Met-p-NA, the kinetic constants k(cat) and K(m) were determined for EcMetAP-I and PfMetAP-II and were compared with those obtained with a "standard" high-performance liquid chromatography (HPLC) discontinuous assay. The assay has also been used to determine the temperature dependence of the kinetic constant k(cat) for PfMetAP-II as well as to screen two novel pseudopeptide inhibitors of MetAPs. The results demonstrate that L-Met-p-NA provides a fast, convenient, and effective substrate for both type I and type II MetAPs and that this substrate can be used to quickly screen inhibitors of MetAPs.     

Beta-aminoketones as prodrugs for selective irreversible inhibitors of type-1 methionine aminopeptidases

We identified and characterized β-aminoketones as prodrugs for irreversible MetAP inhibitors that are selective for the MetAP-1 subtype. β-Aminoketones with certain structural features form α,β-unsaturated ketones under physiological conditions, which bind covalently and selectively to cysteines in the S1 pocket of MetAP-1. The binding mode was confirmed by X-ray crystallography and assays with the MetAPs from Escherichia coli, Staphylococcus aureus and both human isoforms. The initially identified tetralone derivatives showed complete selectivity for E. coli MetAP versus human MetAP-1 and MetAP-2. Rational design of indanone analogs yielded compounds with selectivity for the human type-1 versus the human type-2 MetAP.     

The 1.15A crystal structure of the Staphylococcus aureus methionyl-aminopeptidase and complexes with triazole based inhibitors

Methionyl aminopeptidases (MetAPs) represent a unique class of protease that are responsible for removing the N-terminal methionine residue from proteins and peptides. There are two major classes of MetAPs (type I and type II) described and each class can be subdivided into two subclasses. Eukaryotes contain both the type I and type II MetAPs, whereas prokaryotes possess only the type I enzyme. Due to the physiological importance of these enzymes there is considerable interest in inhibitors to be used as antiangiogenic and antimicrobial agents. Here, we describe the 1.15A crystal structure of the Staphylococcus aureus MetAP-I as an apo-enzyme and its complexes with various 1,2,4-triazole-based derivatives at high-resolution. The protein has a typical "pita-bread" fold as observed for the other MetAP structures. The inhibitors bind in the active site with the N1 and N2 atoms of the triazole moiety complexing two divalent ions. The 1,2,4-triazols represent a novel class of potent non-peptidic inhibitors for the MetAP-Is.     

Characterization of full length and truncated type I human methionine aminopeptidases expressed from Escherichia coli

Methionine aminopeptidase (MetAP) carries out an essential posttranslational modification of nascent proteins by removing the initiator methionine and is recognized as a potential target for developing antibacterial, antifungal, and anticancer agents. We have established an Escherichia coli expression system for human type I MetAP (HsMetAP1) and characterized the full length HsMetAP1 and its N-terminal-truncated mutants HsMetAP1(Delta1-66) and HsMetAP1(Delta1-135) for hydrolysis of several thiopeptolide and peptide substrates and inhibition by a series of nonpeptidic inhibitors. Although the N-terminal extension with zinc finger motifs in HsMetAP1 is not required for enzyme activity, it has a significant impact on the interaction of the enzyme with substrates and inhibitors. In hydrolysis of the thiopeptolide substrates, a relaxation of stringent specificity for the terminal methionine was observed in the truncated mutants. However, this relaxation of specificity was not detectable in hydrolysis of tripeptide or tetrapeptide substrates. Several nonpeptidic inhibitors showed potent inhibition of the mutant HsMetAP1(Delta1-66) but exhibited only weak or no inhibition of the full length enzyme. With the recombinant HsMetAP1 available, we have identified several MetAP inhibitors with submicromolar inhibitory potencies against E. coli MetAP (EcMetAP1) that do not affect HsMetAP1. These results have demonstrated the possibility of developing MetAP inhibitors as antibacterial agents with minimum human toxicity. In addition, micromolar inhibitors of HsMetAP1 identified in this study can serve as tools for investigating the functions of HsMetAP1 in physiological and pathological processes.     

The specificity in vivo of two distinct methionine aminopeptidases in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the essential function of amino-terminal methionine removal is provided cotranslationally by two methionine aminopeptidases (MetAP1 and MetAP2). To examine the individual processing efficiency of each MetAP in vivo, we measured the degree of N-terminal methionine cleavage from a series of mutated glutathione-S-transferase (GST) proteins isolated from yeast wild-type, a map1 deletion strain, a map2 deletion strain, and a map1 deletion strain overexpressing the MAP2 gene. We found that MetAP1 plays the major role in N-terminal methionine removal in yeast. Both MetAPs were less efficient when the second residue was Val, and MetAP2 was less efficient than MetAP1 when the second residue was Gly, Cys, or Thr. These findings indicate that MetAP1 and MetAP2 exhibit different cleavage efficiencies against the same substrates in vivo. Interestingly, although methionine is considered a stabilizing N-terminal residue, we found that retention of the initiator methionine on the Met-Ala-GST mutant protein drastically reduced its half-life in vivo.     

Mutations at the S1 sites of methionine aminopeptidases from Escherichia coli and Homo sapiens reveal the residues critical for substrate specificity

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-Gly-Phe 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.     

Cryo-EM Structures Reveal Relocalization of MetAP in the Presence of Other Protein Biogenesis Factors at the Ribosomal Tunnel Exit

During protein biosynthesis in bacteria, one of the earliest events that a nascent polypeptide chain goes through is the co-translational enzymatic processing. The event includes two enzymatic pathways: deformylation of the N-terminal methionine by the enzyme peptide deformylase (PDF), followed by methionine excision catalyzed by methionine aminopeptidase (MetAP). During the enzymatic processing, the emerging nascent protein likely remains shielded by the ribosome-associated chaperone trigger factor. The ribosome tunnel exit serves as a stage for recruiting proteins involved in maturation processes of the nascent chain. Co-translational processing of nascent chains is a critical step for subsequent folding and functioning of mature proteins.Here, we present cryo-electron microscopy structures of Escherichia coli (E. coli) ribosome in complex with the nascent chain processing proteins. The structures reveal overlapping binding sites for PDF and MetAP when they bind individually at the tunnel exit site, where L22–L32 protein region provides primary anchoring sites for both proteins. In the absence of PDF, trigger factor can access ribosomal tunnel exit when MetAP occupies its primary binding site. Interestingly, however, in the presence of PDF, when MetAP's primary binding site is already engaged, MetAP has a remarkable ability to occupy an alternative binding site adjacent to PDF. Our study, thus, discloses an unexpected mechanism that MetAP adopts for context-specific ribosome association.     

金属离子选择性的甲硫氨酰氨肽酶抑制剂研究进展

甲硫氨酰氨肽酶(MetAP)是潜在的抗细菌、抗真菌和肿瘤治疗的分子靶点。MetAP是一类两价金属离子依赖的蛋白水解酶。然而,生理状态下,MetAP在细胞内结合并利用的金属离子类型目前还没有定论。因而,研究和发展不同金属离子选择性的甲硫氨酰氨肽酶抑制剂对细胞内源性金属离子的解析以及新型抗肿瘤及抗感染药物的研发具有重要意义。     

Structure and function of the methionine aminopeptidases

The removal of the N-terminal methionine from proteins and peptides is dependent upon a novel class of proteases typified by the dinuclear metalloenzyme methionine aminopeptidase from Escherichia coli (eMetAP). Substantial progress has recently been made in determining the structures of several members of this family. The identification of human MetAP as the target of putative anti-cancer drugs reiterates the importance of this family of enzymes. Determination of the modes of binding to E. coli MetAP of a substrate-like bestatin-based inhibitor, as well as phosphorus-containing transition-state analogs and reaction products has led to a rationalization of the substrate specificity and suggested the presumed catalytic mechanism. The conservation of key active site residues and ligand interactions between the MetAPs and other enzyme of the same fold suggest that avoidance of cross-reactivity may be an important consideration in the design of inhibitors directed toward a single member of the family.     

Redox Regulation of Methionine Aminopeptidase 2 Activity

Protein translation is initiated with methionine in eukaryotes, and the majority of proteins have their N-terminal methionine removed by methionine aminopeptidases (MetAP1 and MetAP2) prior to action. Methionine removal can be important for protein function, localization, or stability. No mechanism of regulation of MetAP activity has been identified. MetAP2, but not MetAP1, contains a single Cys²²⁸-Cys⁴⁴⁸ disulfide bond that has an −RHStaple configuration and links two β-loop structures, which are hallmarks of allosteric disulfide bonds. From analysis of crystal structures and using mass spectrometry and activity assays, we found that the disulfide bond exists in oxidized and reduced states in the recombinant enzyme. The disulfide has a standard redox potential of −261 mV and is efficiently reduced by the protein reductant, thioredoxin, with a rate constant of 16,180 m⁻¹ s⁻¹. The MetAP2 disulfide bond also exists in oxidized and reduced states in glioblastoma tumor cells, and stressing the cells by oxygen or glucose deprivation results in more oxidized enzyme. The Cys²²⁸-Cys⁴⁴⁸ disulfide is at the rim of the active site and is only three residues distant from the catalytic His²³¹, which suggested that cleavage of the bond would influence substrate hydrolysis. Indeed, oxidized and reduced isoforms have different catalytic efficiencies for hydrolysis of MetAP2 peptide substrates. These findings indicate that MetAP2 is post-translationally regulated by an allosteric disulfide bond, which controls substrate specificity and catalytic efficiency.     

Molecular discrimination of type-I over type-II methionyl aminopeptidases

Two residues that are conserved in type-I methionyl aminopeptidases (MetAPs) but are absent in all type-II MetAPs are the cysteine residues (Escherichia coli MetAP-I: C59 and C70) that reside at the back of the substrate recognition pocket. These Cys residues are 4.4 A apart and do not form a disulfide bond. Since bacteria and fungi contain only type-I MetAPs while all human cells contain both type-I and type-II MetAPs, type-I MetAPs represent a novel antibiotic/antifungal target if type-I MetAPs can be specifically targeted over type-II. Based on reaction of the thiol-specific binding reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) with the type-I MetAP from E. coli and the type-II MetAP from Pyrococcus furiosus, the type-I MetAP can be selectively inhibited. Verification that DTNB covalently binds to C59 in EcMetAP-I was obtained by mass spectrometry (MS) from reaction of DTNB with the C59A and C70A mutant EcMetAP-I enzymes. In addition, two inhibitors of EcMetAP-I, 5-iodopentaphosphonic acid (1) and 6-phosphonohexanoic acid (2), were designed and synthesized. The first was designed as a selective-C59 binding reagent while the second was designed as a simple competitive inhibitor of EcMetAP. Indeed, inhibitor 1 forms a covalent interaction with C59 based on activity assays and MS measurements, while 2 does not. These data indicate that type-I MetAPs can be selectively targeted over type-II MetAPs, suggesting that type-I MetAPs represent a new enzymatic target for antibacterial or antifungal agents.