The Structure of Human 15-Lipoxygenase-2 with a Substrate Mimic

Kobe, Matthew J.; Neau, David; Mitchell, Caitlin E.; Bartlett, Sue G.; Newcomer, Marcia E.

doi:10.1074/jbc.m113.543777

Cited by 125 publications

(167 citation statements)

References 36 publications

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“…In addition, neither the loopless 15-LOX-2 or 15-LOX-2 in which the Ca 2ϩ binding amino acids had been mutated to Ala revealed this same pattern of distribution upon stimulation (Fig. 7); both variants have wild-type enzymatic properties with free AA as the substrate (10). Only the wild-type enzyme displays an antibody distribution pattern consistent with that of the plasma membrane marker.…”

Section: Cellular Localization Of 15-lox-2-earlier Experiments With 1mentioning

confidence: 72%

“…Enzyme Activity with a Bilayer Mimic-Binding of 15-LOX-2 to nanodiscs was previously demonstrated by analytical size exclusion chromatography (10), and these results established the conditions to investigate membrane-associated enzyme activity with nanodiscs as substrates. The activities of 15-LOX-2, m8S-LOX, and their loop mutants were monitored with AA covalently attached to phospholipids in nanodiscs containing 25% 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (SAPC) as substrate.…”

Section: Resultsmentioning

confidence: 97%

“…Nanodisc Preparation-Nanodiscs were prepared according to previously published protocols (10,39). During the preparation of the phospholipid pellets, the desired ratios of the various sn2 fatty acids and lipid head groups were adjusted.…”

Section: Methodsmentioning

confidence: 99%

“…Cloning was confirmed by sequencing the final pET-Duet-1 vector. The pET-Duet-1 vectors with desired genes were transformed into Rosetta 2 (DE3) cells, and the overexpression was performed as described previously (10).…”

Section: Methodsmentioning

confidence: 99%

“…We recently reported the crystal structure of 15-LOX-2 and described a possible membrane insertion loop and open active site on one face of the elongated protein that would appear to make it feasible for the enzyme to access AA esterified in a membrane phospholipid (PL) (10). We show that in vitro both m8S-LOX and 15-LOX-2 generate the 15-isomer of the product with PL-esterified substrate in a nanodisc membrane bilayer mimic.…”

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confidence: 99%

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Membrane-dependent Activities of Human 15-LOX-2 and Its Murine Counterpart

Bender

Schexnaydre

Murphy

et al. 2016

Journal of Biological Chemistry

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The enzyme encoded by the ALOX15B gene has been linked to the development of atherosclerotic plaques in humans and in a mouse model of hypercholesterolemia. In vitro, these enzymes, which share 78% sequence identity, generate distinct products from their substrate arachidonic acid: the human enzyme, a 15-S-hydroperoxy product; and the murine enzyme, an 8-S-product. We probed the activities of these enzymes with nanodiscs as membrane mimics to determine whether they can access substrate esterified in a bilayer and characterized their activities at the membrane interface. We observed that both enzymes transform phospholipid-esterified arachidonic acid to a 15-S-product. Moreover, when expressed in transfected HEK cells, both enzymes result in significant increases in the amounts of 15-hydroxyderivatives of eicosanoids detected. In addition, we show that 15-LOX-2 is distributed at the plasma membrane when the HEK293 cells are stimulated by the addition Ca 2؉ ionophore and that cellular localization is dependent upon the presence of a putative membrane insertion loop. We also report that sequence differences between the human and mouse enzymes in this loop appear to confer distinct mechanisms of enzyme-membrane interaction for the homologues.A macrophage 2 activity has been linked to elevated levels of oxidized lipids through several experimental approaches that include the heterologous expression of human 15-LOX in a mouse model of hyperlipidemia (1) and pharmacological inhibition of 15-LOX activity (2, 3). These lipid oxidation products can enter the extracellular pool of cholesterol esters transported by LDL (4) and promote the pathological consequences of elevated LDL cholesterol levels (5). Macrophages that take up LDL laden with oxidized lipids are transformed to foam cells, an event that leads to further inflammation and apoptosis and results in the formation of atherosclerotic plaques. Recently, Magnusson et al. (6) demonstrated that silencing production of 15-LOX-2 (the product of the ALOX15B gene) in human macrophages decreased cellular lipid accumulation, the precipitating factor in foam cell formation. These results suggest that inhibition of 15-LOX-2 is a strategy for mitigating the development of cardiovascular disease.Lipoxygenases oxygenate arachidonic acid (AA) to form a stereo-and regiospecific isomer of hydroperoxyeicosatetraenoic acid (HpETE), and isoforms are named according to which carbon atom is oxygenated (for review see Refs. 7 and 8).In general, within a species, lipoxygenases that differ in regiospecificity share ϳ40% sequence identity. In contrast, betweenspecies LOX homologues are expected to share ϳ75% or better sequence identity and generate the same HpETE isomer. The mouse homologue of human 15-LOX-2, however, is an 8-LOX (m8S-LOX) with free AA as the substrate, and this altered regiospecificity appears to be unique to the mouse enzyme (9). We asked whether at the membrane and in a cellular context the mouse enzyme might also generate a 15-HpETE product. This information might prov...

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Section: Cellular Localization Of 15-lox-2-earlier Experiments With 1mentioning

confidence: 72%

Section: Resultsmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Membrane-dependent Activities of Human 15-LOX-2 and Its Murine Counterpart

Bender

Schexnaydre

Murphy

et al. 2016

Journal of Biological Chemistry

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Fatty Acids and their Derivatives

and

Hansen

2016

From Biosynthesis to Total Synthesis

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Lipoxygenase Dynamics and Catalysis Studied by Temperature‐Dependent Hydrogen–Deuterium Exchange

Offenbacher,

Guy

2024

Encyclopedia of Inorganic and Bioinorganic Chemistry

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An active pursuit in biochemistry is the understanding of how the structure and dynamics of protein systems regulate their biological function. In this chapter, we describe temperature‐dependent hydrogen–deuterium exchange mass‐spectrometry (TDHDX‐MS) as a tool used to investigate the structure and dynamics of metalloenzymes in solution. While the method is applicable to many metalloproteins, this review will present TDHDX‐MS studies centered on the widely represented family of mononuclear, non‐heme iron containing lipoxygenase (LOX) enzymes that oxidize fatty acids to initiate biological reactions and activate cellular signaling. We will present how TDHDX has been used to describe the anisotropic nature of energy transfer in LOXs to initiate the rate‐limiting hydrogen atom transfer reaction that proceeds through a tunneling process. Further, TDHDX‐MS has been used detect protein motions and conformations in LOXs related to molecular recognition, protein allostery, and polymorphisms linked to disease.

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The Structure of Human 15-Lipoxygenase-2 with a Substrate Mimic

Cited by 125 publications

References 36 publications

Membrane-dependent Activities of Human 15-LOX-2 and Its Murine Counterpart

Membrane-dependent Activities of Human 15-LOX-2 and Its Murine Counterpart

Fatty Acids and their Derivatives

Lipoxygenase Dynamics and Catalysis Studied by Temperature‐Dependent Hydrogen–Deuterium Exchange

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