David B. Neau

The Structure of Human 5-Lipoxygenase

Substitution of a destabilizing sequence has allowed crystallization of a key enzyme of the inflammatory response. The synthesis of both proinflammatory leukotrienes and anti-inflammatory lipoxins requires the enzyme 5-lipoxygenase (5-LOX). 5-LOX activity is short-lived, apparently in part because of an intrinsic instability of the enzyme. We identified a 5-LOX–specific destabilizing sequence that is involved in orienting the carboxyl terminus, which binds the catalytic iron. Here, we report the crystal structure at 2.4 angstrom resolution of human 5-LOX stabilized by replacement of this sequence.

Structural basis for ligand and heparin binding to neuropilin B domains

Neuropilin (Nrp) is a cell surface receptor with essential roles in angiogenesis and axon guidance. Interactions between Nrp and the positively charged C termini of its ligands, VEGF and semaphorin, are mediated by Nrp domains b1 and b2, which share homology to coagulation factor domains. We report here the crystal structure of the tandem b1 and b2 domains of Nrp-1 (N1b1b2) and show that they form a single structural unit. Cocrystallization of N1b1b2 with Tuftsin, a peptide mimic of the VEGF C terminus, reveals the site of interaction with the basic tail of VEGF on the b1 domain. We also show that heparin promotes N1b1b2 dimerization and map the heparin binding site on N1b1b2. These results provide a detailed picture of interactions at the core of the Nrp signaling complex and establish a molecular basis for the synergistic effects of heparin on Nrp-mediated signaling.

Identification and analysis of a bottleneck in PCB biodegradation.

The microbial degradation of polychlorinated biphenyls (PCBs) provides the potential to destroy these widespread, toxic and persistent environmental pollutants. For example, the four-step upper bph pathway transforms some of the more than 100 different PCBs found in commercial mixtures and is being engineered for more effective PCB degradation. In the critical third step of this pathway, 2,3-dihydroxybiphenyl (DHB) 1,2-dioxygenase (DHBD; EC 1.13.11.39) catalyzes aromatic ring cleavage. Here we demonstrate that ortho-chlorinated PCB metabolites strongly inhibit DHBD, promote its suicide inactivation and interfere with the degradation of other compounds. For example, kcatapp for 2′,6′-diCl DHB was reduced by a factor of ∼7,000 relative to DHB, and it bound with sufficient affinity to competitively inhibit DHB cleavage at nanomolar concentrations. Crystal structures of two complexes of DHBD with ortho-chlorinated metabolites at 1.7 Å resolution reveal an explanation for these phenomena, which have important implications for bioremediation strategies.

The 1.85 A structure of an 8R-lipoxygenase suggests a general model for lipoxygenase product specificity.

Lipoxygenases (LOX) play pivotal roles in the biosynthesis of leukotrienes and other biologically active eicosanoids derived from arachidonic acid. A mechanistic understanding of substrate recognition, when lipoxygenases that recognize the same substrate generate different products, can be used to help guide the design of enzyme-specific inhibitors. We report here the 1.85 A resolution structure of an 8R-lipoxygenase from Plexaura homomalla, an enzyme with a sequence approximately 40% identical to that of human 5-LOX. The structure reveals a U-shaped channel, defined by invariant amino acids, that would allow substrate access to the catalytic iron. We demonstrate that mutations within the channel significantly impact enzyme activity and propose a novel model for substrate binding potentially applicable to other members of this enzyme family.

Conversion of human 5-lipoxygenase to a 15-lipoxygenase by a point mutation to mimic phosphorylation at Serine-663.

The enzyme 5‐lipoxygenase (5‐LOX) initiates biosynthesis of the proinflammatory leukotriene lipid mediators and, together with 15‐LOX, is also required for synthesis of the anti‐inflammatory lipoxins. The catalytic activity of 5‐LOX is regulated through multiple mechanisms, including Ca2+‐targeted membrane binding and phosphorylation at specific serine residues. To investigate the consequences of phosphorylation at S663, we mutated the residue to the phosphorylation mimic Asp, providing a homogenous preparation suitable for catalytic and structural studies. The S663D enzyme exhibits robust 15‐LOX activity, as determined by spectrophotometric and HPLC analyses, with only traces of 5‐LOX activity remaining; synthesis of the anti‐inflammatory lipoxin A4 from arachidonic acid is also detected. The crystal structure of the S663D mutant in the absence and presence of arachidonic acid (in the context of the previously reported Stable‐5‐LOX) reveals substantial remodeling of helices that define the active site so that the once fully encapsulated catalytic machinery is solvent accessible. Our results suggest that phosphorylation of 5‐LOX at S663 could not only down‐regulate leukotriene synthesis but also stimulate lipoxin production in inflammatory cells that do not express 15‐LOX, thus redirecting lipid mediator biosynthesis to the production of proresolving mediators of inflammation.—Gilbert, N. C., Rui, Z., Neau, D. B., Waight, M. T., Bartlett, S. G., Boeglin, W. E., Brash, A. R., Newcomer, M. E. Conversion of human 5‐lipoxygenase to a 15‐lipoxygenase by a point mutation to mimic phosphorylation at Serine‐663. FASEB J. 26, 3222–3229 (2012). www.fasebj.org

The structure of human 15-lipoxygenase-2 with a substrate mimic.

Background: 15-Lipoxygenase-2 is linked to atherosclerotic plaque formation; the homologous enzyme 5-lipoxygenase initiates the synthesis of proinflammatory leukotrienes. Results: We determined the crystal structure of 15-LOX-2 in the presence of a substrate mimic. Conclusion: 15-Lipoxygenase-2 and 5-lipoxgenase display active site variations that confer distinct product specificities. Significance: These differences can be exploited for the design of isoform-specific anti-inflammatories. Atherosclerosis is associated with chronic inflammation occurring over decades. The enzyme 15-lipoxygenase-2 (15-LOX-2) is highly expressed in large atherosclerotic plaques, and its activity has been linked to the progression of macrophages to the lipid-laden foam cells present in atherosclerotic plaques. We report here the crystal structure of human 15-LOX-2 in complex with an inhibitor that appears to bind as a substrate mimic. 15-LOX-2 contains a long loop, composed of hydrophobic amino acids, which projects from the amino-terminal membrane-binding domain. The loop is flanked by two Ca2+-binding sites that confer Ca2+-dependent membrane binding. A comparison of the human 15-LOX-2 and 5-LOX structures reveals similarities at the active sites, as well striking differences that can be exploited for design of isoform-selective inhibitors.

Crystal Structure of a Lipoxygenase in Complex with Substrate THE ARACHIDONIC ACID-BINDING SITE OF 8R-LIPOXYGENASE

Background: Lipoxygenases (LOX) catalyze the oxygenation of polyunsaturated fatty acids but generate distinct products from a common substrate. Results: We report the first structure of a LOX-substrate complex. Conclusion: The structure provides a context for understanding product specificity in enzymes that metabolize arachidonic acid. Significance: With roles in the production of potent lipid mediators, LOX are targets for drug design. Lipoxygenases (LOX) play critical roles in mammalian biology in the generation of potent lipid mediators of the inflammatory response; consequently, they are targets for the development of isoform-specific inhibitors. The regio- and stereo-specificity of the oxygenation of polyunsaturated fatty acids by the enzymes is understood in terms of the chemistry, but structural observation of the enzyme-substrate interactions is lacking. Although several LOX crystal structures are available, heretofore the rapid oxygenation of bound substrate has precluded capture of the enzyme-substrate complex, leaving a gap between chemical and structural insights. In this report, we describe the 2.0 Å resolution structure of 8R-LOX in complex with arachidonic acid obtained under anaerobic conditions. Subtle rearrangements, primarily in the side chains of three amino acids, allow binding of arachidonic acid in a catalytically competent conformation. Accompanying experimental work supports a model in which both substrate tethering and cavity depth contribute to positioning the appropriate carbon at the catalytic machinery.

Directed Evolution of a Ring-cleaving Dioxygenase for Polychlorinated Biphenyl Degradation

DoxG, an extradiol dioxygenase involved in the aerobic catabolism of naphthalene, possesses a weak ability to cleave 3,4-dihydroxybiphenyls (3,4-DHB), critical polychlorinated biphenyl metabolites. A directed evolution strategy combining error-prone PCR, saturation mutagenesis, and DNA shuffling was used to improve the polychlorinated biphenyl-degrading potential of DoxG. Screening was facilitated through analysis of filtered, digital imaging of plated colonies. A simple scheme, which is readily adaptable to other activities, enabled the screening of >105 colonies/h. The best variant, designated DoxGSMA2, cleaved 3,4-DHB with an apparent specificity constant of 2.0 ± 0.3 × 106 m-1 s-1, which is 770 times that of wild-type (WT) DoxG. The specificities of DoxGSMA2 for 1,2-DHN and 2,3-DHB were increased by 6.7-fold and reduced by 2-fold, respectively, compared with the WT enzyme. DoxGSMA2 contained three substituted residues with respect to the WT enzyme: L190M, S191W, and L242S. Structural data indicate that the side chains of residues 190 and 242 occur on opposite walls of the substrate binding pocket and may interact directly with the distal ring of 3,4-DHB or influence contacts between this substrate and other residues. Thus, the introduction of two bulkier residues on one side of the substrate binding pocket and a smaller residue on the other may reshape the binding pocket and alter the catalytically relevant interactions of 3,4-DHB with the enzyme and dioxygen. Kinetic analyses reveal that the substitutions are anti-cooperative.

Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction.

The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nucleolytic ribozymes discovered, the mechanism by which it catalyzes its reaction remains elusive. The nucleobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cleavage site have been observed crystallographically. To better understand how this ribozyme functions, we have solved crystal structures of wild-type and G12A mutant ribozymes. We observe a pH-dependent conformational change centered around G12, consistent with this nucleotide becoming deprotonated. Crystallographic and kinetic analysis of the G12A mutant reveals a Zn(2+) specificity switch suggesting a direct interaction between a divalent metal ion and the purine at position 12. The metal ion specificity switch and the pH-rate profile of the G12A mutant suggest that the minor imino tautomer of A12 serves as the general base in the mutant ribozyme. We propose a model in which the hammerhead ribozyme rearranges prior to the cleavage reaction, positioning two divalent metal ions in the process. The first metal ion, positioned near G12, becomes directly coordinated to the O6 keto oxygen, to lower the pKa of the general base and organize the active site. The second metal ion, positioned near G10.1, bridges the N7 of G10.1 and the scissile phosphate and may participate directly in the cleavage reaction.

Covalent Small Molecule Inhibitors of Ca(2+)-Bound S100B.

Elevated levels of the tumor marker S100B are observed in malignant melanoma, and this EF-hand-containing protein was shown to directly bind wild-type (wt) p53 in a Ca2+-dependent manner, dissociate the p53 tetramer, and inhibit its tumor suppression functions. Likewise, inhibiting S100B with small interfering RNA (siRNAS100B) is sufficient to restore wild-type p53 levels and its downstream gene products and induce the arrest of cell growth and UV-dependent apoptosis in malignant melanoma. Therefore, it is a goal to develop S100B inhibitors (SBiXs) that inhibit the S100B–p53 complex and restore active p53 in this deadly cancer. Using a structure–activity relationship by nuclear magnetic resonance approach (SAR by NMR), three persistent binding pockets are found on S100B, termed sites 1–3. While inhibitors that simultaneously bind sites 2 and 3 are in place, no molecules that simultaneously bind all three persistent sites are available. For this purpose, Cys84 was used in this study as a potential means to bridge sites 1 and 2 because it is located in a small crevice between these two deeper pockets on the protein. Using a fluorescence polarization competition assay, several Cys84-modified S100B complexes were identified and examined further. For five such SBiX–S100B complexes, crystallographic structures confirmed their covalent binding to Cys84 near site 2 and thus present straightforward chemical biology strategies for bridging sites 1 and 3. Importantly, one such compound, SC1982, showed an S100B-dependent death response in assays with WM115 malignant melanoma cells, so it will be particularly useful for the design of SBiX molecules with improved affinity and specificity.