Nathaniel C. Gilbert

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.

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

Structure of a Calcium-dependent 11R-Lipoxygenase Suggests a Mechanism for Ca2+ Regulation

Background: Lipoxygenases vary in their catalytic specificity and regulation. Results: 11R-LOX, strictly Ca2+-dependent, displays novel structural features in the membrane-binding domain. Conclusion: A model for how access to an enclosed active site is linked to Ca2+-dependent membrane binding is proposed. Significance: The 11R-LOX model provides structural insights into the allosteric regulation of lipoxygenases. Lipoxygenases (LOXs) are a key part of several signaling pathways that lead to inflammation and cancer. Yet, the mechanisms of substrate binding and allosteric regulation by the various LOX isoforms remain speculative. Here we report the 2.47-Å resolution crystal structure of the arachidonate 11R-LOX from Gersemia fruticosa, which sheds new light on the mechanism of LOX catalysis. Our crystallographic and mutational studies suggest that the aliphatic tail of the fatty acid is bound in a hydrophobic pocket with two potential entrances. We speculate that LOXs share a common T-shaped substrate channel architecture that gives rise to the varying positional specificities. A general allosteric mechanism is proposed for transmitting the activity-inducing effect of calcium binding from the membrane-targeting PLAT (polycystin-1/lipoxygenase/α-toxin) domain to the active site via a conserved π-cation bridge.

Location, location, location: compartmentalization of early events in leukotriene biosynthesis.

Leukotrienes (LTs), derived from arachidonic acid (AA) released from the membrane by the action of phospholipase A2, are potent lipid mediators of the inflammatory response. In 1983, Dahlén et al. demonstrated that LTC4, LTD4, and LTE4 mediate antigen-induced constriction of bronchi in tissue obtained from subjects with asthma (Dahlén, S. E., Hansson, G., Hedqvist, P., Björck, T., Granström, E., and Dahlén, B. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1712–1716). Over the last 25+ years, substantial progress has been made in understanding how LTs exert their effects, and a broader appreciation for the numerous biological processes they mediate has emerged. LT biosynthesis is initiated by the action of 5-lipoxygenase (5-LOX), which catalyzes the transformation of AA to LTA4 in a two-step reaction. Ca2+ targets 5-LOX to the nuclear membrane, where it co-localizes with the 5-LOX-activating protein FLAP and, when present, the downstream enzyme LTC4 synthase, both transmembrane proteins. Crystal structures of the AA-metabolizing LOXs, LTC4 synthase, and FLAP combined with biochemical data provide a framework for understanding how subcellular organizations optimize the biosynthesis of these labile hydrophobic signaling compounds, which must navigate pathways that include both membrane and soluble enzymes. The insights these structures afford and the questions they engender are discussed in this minireview.

Improving protein crystal quality by selective removal of a Ca2+-dependent membrane-insertion loop

Lipoxygenases (LOXs) catalyze the regiospecific and stereospecific dioxygenation of polyunsaturated membrane-embedded fatty acids. A Ca(2+)-dependent membrane-binding function was localized to the amino-terminal C2-like domain of 8R-lipoxygenase (8R-LOX) from the soft coral Plexaura homomalla. The 3.2 A crystal structure of 8R-LOX and spectroscopic data suggested that Ca(2+) stabilizes two membrane-insertion loops. Analysis of the protein packing contacts in the crystal lattice indicated that the conformation of one of the two loops complicated efforts to improve the resolution of the X-ray data. A deletion mutant of 8R-LOX in which the corresponding membrane-insertion loop is absent (Delta41-45:GSLOX) was engineered. Removal of the membrane-insertion loop dramatically increases the protein yield from bacterial cultures and the quality of the crystals obtained, resulting in a better than 1 A improvement in the resolution of the diffraction data.

A covalent linker allows for membrane targeting of an oxylipin biosynthetic complex.

A naturally occurring bifunctional protein from Plexaura homomalla links sequential catalytic activities in an oxylipin biosynthetic pathway. The C-terminal lipoxygenase (LOX) portion of the molecule catalyzes the transformation of arachidonic acid (AA) to the corresponding 8 R-hydroperoxide, and the N-terminal allene oxide synthase (AOS) domain promotes the conversion of the hydroperoxide intermediate to the product allene oxide (AO). Small-angle X-ray scattering data indicate that in the absence of a covalent linkage the two catalytic domains that transform AA to AO associate to form a complex that recapitulates the structure of the bifunctional protein. The SAXS data also support a model for LOX and AOS domain orientation in the fusion protein inferred from a low-resolution crystal structure. However, results of membrane binding experiments indicate that covalent linkage of the domains is required for Ca (2+)-dependent membrane targeting of the sequential activities, despite the noncovalent domain association. Furthermore, membrane targeting is accompanied by a conformational change as monitored by specific proteolysis of the linker that joins the AOS and LOX domains. Our data are consistent with a model in which Ca (2+)-dependent membrane binding relieves the noncovalent interactions between the AOS and LOX domains and suggests that the C2-like domain of LOX mediates both protein-protein and protein-membrane interactions.