Crystal Structure of a Lipoxygenase in Complex with Substrate

Neau, David; Bender, Güneş; Boeglin, William E.; Bartlett, Sue G.; Brash, Alan R.; Newcomer, Marcia E.

doi:10.1074/jbc.m114.599662

Cited by 62 publications

(53 citation statements)

References 44 publications

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“…Extrapolating from the structure of 8 R -LOX in complex with AA (23) , His-600 is deep in the active site where the tubular cavity dead-ends (Fig 1, 2). We tested whether substitution of this charged/polar amino acid with a hydrophobic amino acid might impact 5-LOX activity with two mutations: H600A and H600V.…”

Section: Resultsmentioning

confidence: 99%

“…Mutations were made to Phe-177, Tyr-181, Ala-603, and Trp-147. The first three cluster to truncate one arm of what has been described as a U-shaped cavity in homologous enzymes with accessible catalytic sites (17) (15-LOX types-1 (19) and -2 (20) , 12-LOX (21) and 8 R -LOX (22, 23) . However, the St-5-LOX structure revealed that Phe-177 and Tyr-181 form what has been described as an active site “cork” and the small size of the Ala at 603 appears to allow insertion of the “cork” into the active site.…”

Section: Resultsmentioning

confidence: 99%

“…Lastly, H600, positioned innermost in the tubular, U-shaped cavity that accommodates the methyl end of arachidonic acid in the 8 R -LOX:AA complex structure (17, 23) , was mutated to a hydrophobic amino acid to test whether it is important for “head first” binding.…”

Section: Resultsmentioning

confidence: 99%

“…Thus “removal” of the bulky Tyr is consistent with an enhanced entry and/or egress from the active site. (For comparison, the values of k cat for lipoxygenases with open-access sites can vary from ~1 s −1 to ~200 s −1 (15-LOX-2 (20) and 8- R -LOX (23) ). The exceptional activity of the 8 R -enzyme is likely a consequence of an O 2 access channel that is open to solvent.)…”

Section: Resultsmentioning

confidence: 99%

“…Several LOX structures are available and combine to provide details of a consensus active site (17) , but the 5-LOX isoform differs significantly from 15-LOX-1 (18, 19) , 15-LOX-2 (20) , 12-LOX (21) and 8 R -LOX (22, 23) in three important ways: (1) The HPETE produced by 5-LOX is an intermediate; a second reaction in the same 5-LOX active site converts the intermediate to LTA 4 . (However, in vitro 15-LOX-1 can also utilize 15-HPETE as a substrate and generate a product analogous to LTA 4 (24) ).…”

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confidence: 99%

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Identification of the Substrate Access Portal of 5-Lipoxygenase

2015

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The overproduction of inflammatory lipid mediators derived from arachidonic acid contributes to asthma and cardiovascular diseases, among other pathologies. Consequently, the enzyme that initiates the synthesis of pro-inflammatory leukotrienes, 5-lipoxygenase (5-LOX), is a target for drug design. The crystal structure of 5-LOX revealed a fully encapsulated active site, thus the point of substrate entry is not known. We asked whether a structural motif, a “cork” present in 5-LOX but absent in other mammalian lipoxygenases, might be ejected to allow substrate access. Our results indicate that reduction of cork volume facilitates access to the active site. However, if cork entry into the site is obstructed, enzyme activity is significantly compromised. The results support a model in which the “cork” that shields the active site in the absence of substrate serves as the active site portal, but the “corking” amino acid Phe-177 plays a critical role in providing a fully functional active site. Thus the more appropriate metaphor for this structural motif is a “twist-and-pour” cap. Additional mutagenesis data are consistent with a role for His-600, deep in the elongated cavity, in positioning the substrate for catalysis.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Identification of the Substrate Access Portal of 5-Lipoxygenase

2015

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EPR Spectroscopic Studies of Lipoxygenases

Gaffney

2019

Chemistry An Asian Journal

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Polyunsaturated fatty acids are sources of diverse natural, and chemically designed products. The enzyme lipoxygenase selectively oxidizes fatty acid acyl chains using controlled free radical chemistry; the products are regio‐ and stereo‐chemically unique hydroperoxides. A conserved structural fold of ≈600 amino acids harbors a long and narrow substrate channel and a well‐shielded catalytic iron. Oxygen, a co‐substrate, is blocked from the active site until a hydrogen atom is abstracted from substrate bis‐allylic carbon, in a non‐heme iron redox cycle. EPR spectroscopy of ferric intermediates in lipoxygenase catalysis reveals changes in the metal coordination and leads to a proposal on the nature of the reactive intermediate. Remarkably, free radicals are so well controlled in lipoxygenase chemistry that spin label technology can be applied as well. The current level of understanding of steps in lipoxygenase catalysis, from the EPR perspective, will be reviewed.

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The structural basis for specificity in lipoxygenase catalysis

2015

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Many intriguing facets of lipoxygenase (LOX) catalysis are open to a detailed structural analysis. Polyunsaturated fatty acids with two to six double bonds are oxygenated precisely on a particular carbon, typically forming a single chiral fatty acid hydroperoxide product. Molecular oxygen is not bound or liganded during catalysis, yet it is directed precisely to one position and one stereo configuration on the reacting fatty acid. The transformations proceed upon exposure of substrate to enzyme in the presence of O 2 (RH 1 O 2 fi ROOH), so it has proved challenging to capture the precise mode of substrate binding in the LOX active site. Beginning with crystal structures with bound inhibitors or surrogate substrates, and most recently arachidonic acid bound under anaerobic conditions, a picture is consolidating of catalysis in a U-shaped fatty acid binding channel in which individual LOX enzymes use distinct amino acids to control the head-to-tail orientation of the fatty acid and register of the selected pentadiene opposite the non-heme iron, suitably positioned for the initial stereoselective hydrogen abstraction and subsequent reaction with O 2 . Drawing on the crystal structures available currently, this review features the roles of the N-terminal bbarrel (C2-like, or PLAT domain) in substrate acquisition and sensitivity to cellular calcium, and the a-helical catalytic domain in fatty acid binding and reactions with O 2 that produce hydroperoxide products with regio and stereospecificity. LOX structures combine to explain how similar enzymes with conserved catalytic machinery differ in product, but not substrate, specificities.

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Crystal Structure of a Lipoxygenase in Complex with Substrate

Cited by 62 publications

References 44 publications

Identification of the Substrate Access Portal of 5-Lipoxygenase

Identification of the Substrate Access Portal of 5-Lipoxygenase

EPR Spectroscopic Studies of Lipoxygenases

The structural basis for specificity in lipoxygenase catalysis

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