A comprehensive model of positional and stereo control in lipoxygenases

Coffa, Gianguido; Schneider, Claus; Brash, Alan R.

doi:10.1016/j.bbrc.2005.07.185

Cited by 90 publications

(99 citation statements)

References 42 publications

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“…HPLC-purified primers were purchased from Eurofins MWG Operon, and sequencing was performed by UC Berkeley Sequencing Facility. The V564F lipoxygenase mutant was expressed and purified using the SLO pT7-7 plasmid in Escherichia coli, as described previously (29,31). WT SLO and all other mutants were expressed and purified as His tag fusion proteins using the SLO pET-30Xa/LIC plasmid, acquired from Professor Betty Gaffney at Florida State University.…”

Section: Methodsmentioning

confidence: 99%

“…WT SLO and all other mutants were expressed and purified as His tag fusion proteins using the SLO pET-30Xa/LIC plasmid, acquired from Professor Betty Gaffney at Florida State University. These enzymes were expressed and purified as described previously, with several modifications (31). His-tagged SLO was eluted with wash buffer containing 250 mM imidazole and dialyzed overnight (4°C) into 20 mM Bis-Tris (pH 6.6) buffer.…”

Section: Methodsmentioning

confidence: 99%

“…Enzyme Kinetics-Oxygen electrode and UV kinetic assays were performed for WT SLO and each mutant lipoxygenase as described previously (19,29,31). Oxygen concentration was varied from 30 to 1365 M. LA concentration was varied from 2 to 80 M. All kinetic assays were performed at 20°C and in 0.1 M borate (pH 9.0).…”

Section: Methodsmentioning

confidence: 99%

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Control of the Position of Oxygen Delivery in Soybean Lipoxygenase-1 by Amino Acid Side Chains within a Gas Migration Channel

Collazo

Klinman

2016

Journal of Biological Chemistry

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Understanding gas migration pathways is critical to unraveling structure-function relationships in enzymes that process gaseous substrates such as O 2 , H 2 , and N 2 . This work investigates the role of a defined pathway for O 2 in regulating the peroxidation of linoleic acid by soybean lipoxygenase 1. Computational and mutagenesis studies provide strong support for a dominant delivery channel that shuttles molecular oxygen to a specific region of the active site, thereby ensuring the regio-and stereospecificity of product. Analysis of reaction kinetics and product distribution in channel mutants also reveals a plasticity to the gas migration pathway. The findings show that a single site mutation (I553W) limits oxygen accessibility to the active site, greatly increasing the fraction of substrate that reacts with oxygen free in solution. They also show how a neighboring site mutation (L496W) can result in a redirection of oxygen toward an alternate position of the substrate, changing the regio-and stereospecificity of peroxidation. The present data indicate that modest changes in a protein scaffold may modulate the access of small gaseous molecules to enzyme-bound substrates.Structure-function studies of enzymes have moved beyond an exclusive focus on active site protein side chains, with the growing recognition that distal residues can have a significant impact on catalytic bond cleavage events (1, 2). Topological features within the protein matrix, such as cavities and channels, can also play key roles in the flux of biomolecules. For proteins with gaseous substrates, e.g. O 2 , H 2 , N 2 , and NH 3 , specific channels have been proposed either to transport reactive intermediates between active sites within a multifunctional enzyme (3) or to guide these small molecules from the solvent to the active site. The likely importance of the latter is relevant to a wide range of enzymes that includes oxidases, monooxygenases, nitrogenases, and hydrogenases (4 -17). The involvement of functionally evolved gas channels represents a significant departure from earlier held views in which random, transient fluctuations within a protein were proposed to facilitate the delivery of gaseous substrates to their site of binding and/or reactivity.Studies of lipoxygenases have aided in this shift in perception, given their requirement to capture O 2 via highly regio-and stereospecific pathways. As illustrated in Fig. 1 for the reaction catalyzed by soybean lipoxygenase-1 (SLO-1), 3 the delocalized nature of the free radical intermediate generated from the preferred substrate linoleic acid (LA) predicts that four unique products will be produced in equal amounts in solution. By contrast, native SLO-1 produces the 13S-hydroperoxide product (13S-HPOD) in greater than 90% yield for reaction of WT enzyme under optimal conditions. The precise mechanism by which SLO-1, as well as other lipoxygenases, maintain the regio-and stereospecificity of product peroxidation has engendered a variety of proposals that include: (i) an alteration in s...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Control of the Position of Oxygen Delivery in Soybean Lipoxygenase-1 by Amino Acid Side Chains within a Gas Migration Channel

Collazo

Klinman

2016

Journal of Biological Chemistry

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“…The molecular determinants of stereo specificity of the LOX reaction have been the focus of a number of recent studies [6]. These studies have highlighted the importance of a single amino acid residue, which is a conserved alanine in S-specific LOX and a glycine in the case of R-LOX.…”

Section: Introductionmentioning

confidence: 99%

On the Substrate Binding of Linoleate 9‐Lipoxygenases

Andreou

Hornung

Kunze

et al. 2008

Lipids

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Lipoxygenases (LOX; linoleate:oxygen oxidoreductase EC 1.13.11.12) consist of a class of enzymes that catalyze the regio-and stereo specific dioxygenation of polyunsaturated fatty acids. Here we characterize two proteins that belong to the less studied class of 9-LOXs, Solanum tuberosum StLOX1 and Arabidopsis thaliana At-LOX1. The proteins were recombinantly expressed in E. coli and the product specificity of the enzymes was tested against different fatty acid substrates. Both enzymes showed high specificity against all tested C18 fatty acids and produced (9S)-hydroperoxides. However, incubation of the C20 fatty acid arachidonic acid with AtLOX1 gave a mixture of racemic hydroperoxides. On the other hand, with StLOX1 we observed the formation of a mixture of products among which the (5S)-hydroperoxy eicosatetraenoic acid (5S-H(P)ETE) was the most abundant. Esterified fatty acids were no substrates. We used site directed mutagenesis to modify a conserved valine residue in the active site of StLOX1 and examine the importance of space within the active site, which has been shown to play a role in determining the positional specificity. The Val576Phe mutant still catalyzed the formation of (9S)-hydroperoxides with C18 fatty acids, while it exhibited altered specificity against arachidonic acid and produced mainly (11S)-H(P)ETE. These data confirm the model that in case of linoleate 9-LOX binding of the substrate takes place with the carboxyl-group first.

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“…Metabolism of PC Esters-The ability of a LOX enzyme to utilize esterified fatty acids with a bulky head group (such as PC esters) as substrate has been implicated with the substrate binding orientation. In the case of a LOX boot-shaped binding site hypothesis it is assumed that a LOX reaction with such substrates is only possible, when fatty acid binding takes place with the methyl end first (38). For this reason we investigated the metabolism of PC-20:4(n-6) by CspLOX2.…”

mentioning

confidence: 99%

A Bisallylic Mini-lipoxygenase from Cyanobacterium Cyanothece sp. That Has an Iron as Cofactor

Andreou¹,

Göbel²,

Hámberg

et al. 2010

Journal of Biological Chemistry

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Lipoxygenases are enzymes that are found ubiquitously in higher animals and plants, but have only recently been identified in a number of bacteria. The genome of the diazotrophic unicellular cyanobacterium Cyanothece sp. harbors two genes with homology to lipoxygenases. Here we describe the isolation of one gene, formerly named csplox2. It was cloned, and the protein was expressed in Escherichia coli and purified. The purified enzyme belongs to the group of prokaryotic mini lipoxygenases, because it had a molecular mass of 65 kDa. Interestingly, it catalyzed the conversion of linoleic acid, the only endogenously found polyunsaturated fatty acid, primarily to the bisallylic hydroperoxide 11R-hydroperoxyoctadecadienoic acid. This product had previously only been described for the manganese lipoxygenase from the take all fungus, Gaeumannomyces graminis. By contrast, CspLOX2 was shown to be an iron lipoxygenase. In addition, CspLOX2 formed a mixture of typical conjugated lipoxygenase products, e.g. 9R-and 13S-hydroperoxide. The conversion of linoleic acid took place with a maximum reaction rate of 31 s ؊1 . Incubation of the enzyme with [(11S)-2 H]linoleic acid led to the formation of hydroperoxides that had lost the deuterium label, thus suggesting that CspLOX2 catalyzes antarafacial oxygenation as opposed to the mechanism of manganese lipoxygenase. CspLOX2 could also oxidize diarachidonylglycerophosphatidylcholine with similar specificity as the free fatty acid, indicating that binding of the substrate takes place with a "tail-first" orientation. We conclude that CspLOX2 is a novel iron mini-lipoxygenase that catalyzes the formation of bisallylic hydroperoxide as the major product.

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A comprehensive model of positional and stereo control in lipoxygenases

Cited by 90 publications

References 42 publications

Control of the Position of Oxygen Delivery in Soybean Lipoxygenase-1 by Amino Acid Side Chains within a Gas Migration Channel

Control of the Position of Oxygen Delivery in Soybean Lipoxygenase-1 by Amino Acid Side Chains within a Gas Migration Channel

On the Substrate Binding of Linoleate 9‐Lipoxygenases

A Bisallylic Mini-lipoxygenase from Cyanobacterium Cyanothece sp. That Has an Iron as Cofactor

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