Fredrik Tholander

Structure-based dissection of the active site chemistry of leukotriene a4 hydrolase: implications for m1 aminopeptidases and inhibitor design.

M1 aminopeptidases comprise a large family of biologically important zinc enzymes. We show that peptide turnover by the M1 prototype, leukotriene A4 hydrolase/aminopeptidase, involves a shift in substrate position associated with exchange of zinc coordinating groups, while maintaining the overall coordination geometry. The transition state is stabilized by residues conserved among M1 members and in the final reaction step, Glu-296 of the canonical zinc binding HEXXH motif shuffles a proton from the hydrolytic water to the leaving group. Tripeptide substrates bind along the conserved GXMEN motif, precisely occupying the distance between Glu-271 and Arg-563, whereas the Arg specificity is governed by a narrow S1 pocket capped with Asp-375. Our data provide detailed insights to the active site chemistry of M1 aminopeptidases and will aid in the development of novel enzyme inhibitors.

Leukotriene A4 hydrolase.

The leukotrienes (LTs) are a family of lipid mediators involved in inflammation and allergy. Leukotriene B4 is a classical chemoattractant, which triggers adherence and aggregation of leukocytes to the endothelium at only nanomolar concentrations. In addition, leukotriene B4 modulates immune responses, participates in the host-defense against infections, and is a key mediator of PAF-induced lethal shock. Because of these powerful biological effects, leukotriene B4 is implicated in a variety of acute and chronic inflammatory diseases, e.g. nephritis, arthritis, dermatitis, and chronic obstructive pulmonary disease. The final step in the biosynthesis of leukotriene B4 is catalyzed by leukotriene A4 hydrolase, a unique bi-functional zinc metalloenzyme with an anion-dependent aminopeptidase activity. Here we describe the most recent developments regarding our understanding of the structure, function, and catalytic mechanisms of leukotriene A4 hydrolase.

VEGF-B promotes cancer metastasis through a VEGF-A–independent mechanism and serves as a marker of poor prognosis for cancer patients

Significance Cancer metastasis is responsible for a majority of the mortality in cancer patients and involves complex interactions, modulated by various factors and cytokines, between malignant and host cells. Vascular structures in solid tumors are crucial for cancer cell intravasation into the circulation. Our present work shows that VEGF-B produced by tumor cells significantly remodels tumor microvasculature, leading to leaky vascular networks that are highly permissive for tumor cell invasion. VEGF-B–promoted cancer metastasis occurs through a VEGF-A–independent mechanism. Thus, inhibition of VEGF-B should be considered an independent approach for the development of new drugs for the treatment of cancer invasion and metastasis. VEGF-B also may be considered as an independent prognostic marker for cancer metastasis. The biological functions of VEGF-B in cancer progression remain poorly understood. Here, we report that VEGF-B promotes cancer metastasis through the remodeling of tumor microvasculature. Knockdown of VEGF-B in tumors resulted in increased perivascular cell coverage and impaired pulmonary metastasis of human melanomas. In contrast, the gain of VEGF-B function in tumors led to pseudonormalized tumor vasculatures that were highly leaky and poorly perfused. Tumors expressing high levels of VEGF-B were more metastatic, although primary tumor growth was largely impaired. Similarly, VEGF-B in a VEGF-A–null tumor resulted in attenuated primary tumor growth but substantial pulmonary metastases. VEGF-B also led to highly metastatic phenotypes in Vegfr1 tk−/− mice and mice treated with anti–VEGF-A. These data indicate that VEGF-B promotes cancer metastasis through a VEGF-A–independent mechanism. High expression levels of VEGF-B in two large-cohort studies of human patients with lung squamous cell carcinoma and melanoma correlated with poor survival. Taken together, our findings demonstrate that VEGF-B is a vascular remodeling factor promoting cancer metastasis and that targeting VEGF-B may be an important therapeutic approach for cancer metastasis.

Leukotriene A4 hydrolase: Selective abrogation of leukotriene B4 formation by mutation of aspartic acid 375

Leukotriene A4 (LTA4, 5S-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid) hydrolase (LTA4H)/aminopeptidase is a bifunctional zinc metalloenzyme that catalyzes the final and rate-limiting step in the biosynthesis of leukotriene B4 (LTB4, 5S,12R-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid), a classical chemoattractant and immune modulating lipid mediator. Two chemical features are key to the bioactivity of LTB4, namely, the chirality of the 12R-hydroxyl group and the cis-trans-trans geometry of the conjugated triene structure. From the crystal structure of LTA4H, a hydrophilic patch composed of Gln-134, Tyr-267, and Asp-375 was identified in a narrow and otherwise hydrophobic pocket, believed to bind LTA4. In addition, Asp-375 belongs to peptide K21, a previously characterized 21-residue active site-peptide to which LTA4 binds during suicide inactivation. In the present report we used site-directed mutagenesis and x-ray crystallography to show that Asp-375, but none of the other candidate residues, is specifically required for the epoxide hydrolase activity of LTA4H. Thus, mutation of Asp-375 leads to a selective loss of the enzymes ability to generate LTB4 whereas the aminopeptidase activity is preserved. We propose that Asp-375, possibly assisted by Gln-134, acts as a critical determinant for the stereoselective introduction of the 12R-hydroxyl group and thus the biological activity of LTB4.

Discovery of antimicrobial ribonucleotide reductase inhibitors by screening in microwell format

Ribonucleotide reductase (RNR) catalyzes reduction of the four different ribonucleotides to their corresponding deoxyribonucleotides and is the rate-limiting enzyme in DNA synthesis. RNR is a well-established target for the antiproliferative drugs Gemzar and Hydrea, for antisense therapy, and in combination chemotherapies. Surprisingly, few novel drugs that target RNR have emerged, partly because RNR activity assays are laboratory-intense and exclude high-throughput methodologies. Here, we present a previously undescribed PCR-based assay for RNR activity measurements in microplate format. We validated the approach by screening a diverse library of 1,364 compounds for inhibitors of class I RNR from the opportunistic pathogen Pseudomonas aeruginosa, and we identified 27 inhibitors with IC50 values from ∼200 nM to 30 μM. Interestingly, a majority of the identified inhibitors have been found inactive in human cell lines as well as in anticancer and in vivo tumor tests as reported by the PubChem BioAssay database. Four of the RNR inhibitors inhibited growth of P. aeruginosa, and two were also found to affect the transcription of RNR genes and to decrease the cellular deoxyribonucleotide pools. This unique PCR-based assay works with any RNR enzyme and any substrate nucleotide, and thus opens the door to high-throughput screening for RNR inhibitors in drug discovery.

Crystal structure of leukotriene A4 hydrolase in complex with kelatorphan, implications for design of zinc metallopeptidase inhibitors.

Leukotriene A4 hydrolase (LTA4H) is a key enzyme in the inflammatory process of mammals. It is an epoxide hydrolase and an aminopeptidase of the M1 family of the MA clan of Zn‐metallopeptidases. We have solved the crystal structure of LTA4H in complex with N‐[3(R)‐[(hydroxyamino)carbonyl]‐2‐benzyl‐1‐oxopropyl]‐L‐alanine, a potent inhibitor of several Zn‐metalloenzymes, both endopeptidases and aminopeptidases. The inhibitor binds along the sequence signature for M1 aminopeptidases, GXMEN. It exhibits bidentate chelation of the catalytic zinc and binds to LTA4Hs enzymatically essential carboxylate recognition site. The structure gives clues to the binding of this inhibitor to related enzymes and thereby identifies residues of their S1′ sub sites as well as strategies for design of inhibitors.

Biochemical Characterization of the Split Class II Ribonucleotide Reductase from Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa can grow under both aerobic and anaerobic conditions. Its flexibility with respect to oxygen load is reflected by the fact that its genome encodes all three existing classes of ribonucleotides reductase (RNR): the oxygen-dependent class I RNR, the oxygen-indifferent class II RNR, and the oxygen-sensitive class III RNR. The P. aeruginosa class II RNR is expressed as two separate polypeptides (NrdJa and NrdJb), a unique example of a split RNR enzyme in a free-living organism. A split class II RNR is also found in a few closely related γ-Proteobacteria. We have characterized the P. aeruginosa class II RNR and show that both subunits are required for formation of a biologically functional enzyme that can sustain vitamin B12-dependent growth. Binding of the B12 coenzyme as well as substrate and allosteric effectors resides in the NrdJa subunit, whereas the NrdJb subunit mediates efficient reductive dithiol exchange during catalysis. A combination of activity assays and activity-independent methods like surface plasmon resonance and gas phase electrophoretic macromolecule analysis suggests that the enzymatically active form of the enzyme is a (NrdJa-NrdJb)2 homodimer of heterodimers, and a combination of hydrogen-deuterium exchange experiments and molecular modeling suggests a plausible region in NrdJa that interacts with NrdJb. Our detailed characterization of the split NrdJ from P. aeruginosa provides insight into the biochemical function of a unique enzyme known to have central roles in biofilm formation and anaerobic growth.

The Crystal Structure of Thermotoga Maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), alone and in several complexes, including with the allosteric effector dATP and its cognate substrate CTP. In the crystal structure of the enzyme as purified, tmNrdD lacks a cysteine for radical transfer to the substrate pre-positioned in the active site. Nevertheless activity assays using anaerobic cell extracts from T. maritima demonstrate that the class III RNR is enzymatically active. Other genetic and microbiological evidence is summarized indicating that the enzyme is important for T. maritima. Mutation of either of two cysteine residues in a disordered loop far from the active site results in inactive enzyme. We discuss the possible mechanisms for radical initiation of substrate reduction given the collected evidence from the crystal structure, our activity assays and other published work. Taken together, the results suggest either that initiation of substrate reduction may involve unprecedented conformational changes in the enzyme to bring one of these cysteine residues to the expected position, or that alternative routes for initiation of the RNR reduction reaction may exist. Finally, we present a phylogenetic analysis showing that the structure of tmNrdD is representative of a new RNR subclass IIIh, present in all Thermotoga species plus a wider group of bacteria from the distantly related phyla Firmicutes, Bacteroidetes and Proteobacteria.

Assay for rapid analysis of the tri-peptidase activity of LTA4 hydrolase

Leukotriene A4 hydrolase is a bifunctional zinc metalloenzyme with an epoxide hydrolase activity as well as an arginyl tri‐peptidase activity. Detailed enzymological and mechanistic investigations of the latter activity have been hampered by the lack of a rapid and convenient enzyme assay. Here we have developed a new method allowing direct spectrophotometric assessment of the tri‐peptide cleaving activity of leukotriene A4 hydrolase, as well as other peptidases. The method utilizes two competing substrates, one chromogenic reference substrate together with the tri‐peptide substrate of interest, and relies on computer‐assisted analysis of progress curves. The chromogenic reference substrate serves to disclose the “invisible” tri‐peptide substrate for kinetic analysis. The method is fast and simple and will allow detailed kinetic studies and screening for natural peptide substrates of leukotriene A4 hydrolase as well as other members of the M1 family of aminopeptidases. Proteins 2007.

Phosphorylation of Leukotriene C4 Synthase at Serine 36 Impairs Catalytic Activity.

Leukotriene C4 synthase (LTC4S) catalyzes the formation of the proinflammatory lipid mediator leukotriene C4 (LTC4). LTC4 is the parent molecule of the cysteinyl leukotrienes, which are recognized for their pathogenic role in asthma and allergic diseases. Cellular LTC4S activity is suppressed by PKC-mediated phosphorylation, and recently a downstream p70S6k was shown to play an important role in this process. Here, we identified Ser36 as the major p70S6k phosphorylation site, along with a low frequency site at Thr40, using an in vitro phosphorylation assay combined with mass spectrometry. The functional consequences of p70S6k phosphorylation were tested with the phosphomimetic mutant S36E, which displayed only about 20% (20 μmol/min/mg) of the activity of WT enzyme (95 μmol/min/mg), whereas the enzyme activity of T40E was not significantly affected. The enzyme activity of S36E increased linearly with increasing LTA4 concentrations during the steady-state kinetics analysis, indicating poor lipid substrate binding. The Ser36 is located in a loop region close to the entrance of the proposed substrate binding pocket. Comparative molecular dynamics indicated that Ser36 upon phosphorylation will pull the first luminal loop of LTC4S toward the neighboring subunit of the functional homotrimer, thereby forming hydrogen bonds with Arg104 in the adjacent subunit. Because Arg104 is a key catalytic residue responsible for stabilization of the glutathione thiolate anion, this phosphorylation-induced interaction leads to a reduction of the catalytic activity. In addition, the positional shift of the loop and its interaction with the neighboring subunit affect active site access. Thus, our mutational and kinetic data, together with molecular simulations, suggest that phosphorylation of Ser36 inhibits the catalytic function of LTC4S by interference with the catalytic machinery.