Purification and Catalytic Activities of the Two Domains of the Allene Oxide Synthase-Lipoxygenase Fusion Protein of the CoralPlexaura homomalla

Boutaud, Olivier; Brash, Alan R.

doi:10.1074/jbc.274.47.33764

Cited by 70 publications

(104 citation statements)

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“…During these reactions, cAOS inactivates irreversibly after tens of seconds (7). Although the proposed mechanism of allene oxide synthases involves homolytic cleavage of hydroperoxide, many hemoproteins react with organic hydroperoxides by a mixture of homolysis and heterolysis (18).…”

Section: Discussion Stoichiometric Oxidation Of Coral Aos By Peracetimentioning

confidence: 99%

“…HpETE) in the intact fusion protein, as well as when it is expressed separately, but it does not exhibit catalase activity (7). Evidence from sequence and spectroscopy has confirmed that the similarity of coral AOS to catalase includes tyrosinate axial ligation to heme iron (6,8).…”

mentioning

confidence: 99%

“…It is interesting that coral AOS inactivates rapidly, after turning over only ~32000 molecules of substrate (7). Inactivation of cAOS could occur if occasional heterolytic cleavage of the hydroperoxide substrate results in overoxidation of the heme or the protein.…”

mentioning

confidence: 99%

“…In some catalases, the subsequent transfer of an electron from tyrosine to the porphyrin π-cation radical of compound I gives intermediate II, with an iron oxyferryl and a protein-bound tyrosine radical (20)(21)(22)(23). Since cAOS does not react readily with hydrogen peroxide, and it reacts very rapidly (1400 turnovers s −1 ) with its natural substrate, 8R-hydroperoxyeicosatetraenoic acid (8R-HpETE) (7), it has been of interest to examine whether there are reactions of an intermediate rate, with other hydroperoxides, that might allow cAOS intermediates to be trapped. We reported that cAOS, like bovine liver catalase (BLC), reacts with peracetic acid to form a protein radical with features in the EPR spectrum suggesting tyrosyl as the origin (8).…”

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Role of Radical Formation at Tyrosine 193 in the Allene Oxide Synthase Domain of a Lipoxygenase−AOS Fusion Protein from Coral

Katsir

Seavy

et al. 2003

Biochemistry

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Coral allene oxide synthase (cAOS), a fusion protein with 8R-lipoxygenase in Plexaura homomalla, is a hemoprotein with sequence similarity to catalases. cAOS reacts rapidly with the oxidant peracetic acid to form heme compound I and intermediate II. Concomitantly, an electron paramagnetic resonance (EPR) signal with tyrosyl radical-like features, centered at a g-value of 2.004-2.005, is formed. The radical is identified as tyrosyl by changes in EPR spectra when deuterated tyrosine is incorporated in cAOS. The radical location in cAOS is determined by mutagenesis of Y193 and Y209. Upon oxidation, native cAOS and mutant Y209F exhibit the same radical spectrum, but no significant tyrosine radical forms in mutant Y193H, implicating Y193 as the radical site in native cAOS. Estimates of the side chain torsion angles for the radical at Y193, based on the β-proton isotropic EPR hyperfine splitting, A iso , are θ 1 = 21 to 30° and θ 2 = −99 to −90°. The results show that cAOS can cleave nonsubstrate hydroperoxides by a heterolytic path, although a homolytic course is likely taken in converting the normal substrate, 8R-hydroperoxyeicosatetraenoic acid (8R-HpETE), to product. Coral AOS achieves specificity for the allene oxide formed by selection of the homolytic pathway normally, while it inactivates by the heterolytic path with nonoptimal substrates. Accordingly, with the nonoptimal substrate, 13R-hydroperoxyoctadecadienoic acid (13R-HpODE), mutant Y193H is inactivated after turning over significantly fewer substrate molecules than required to inactivate native cAOS or the Y209F mutant because it cannot absorb oxidizing equivalents by forming a radical at Y193. Polyunsaturated fatty acids (PUFAs) 1 are rapidly converted to cell signaling molecules in multistep syntheses involving an initial lipoxygenase or prostaglandin synthase step, followed by further enzymatic conversions (1-3). These multistep syntheses are regulated by localization of the enzymes in subcellular compartments and by protein-protein associations. Examples include synthesis of leukotriene C 4 at the nuclear envelope of leukocytes (4) and synthesis of jasmonic acid by steps which are thought to begin in chloroplasts and to be completed in peroxisomes of green plant cells (5). A fusion protein identified in the coral Plexaura homomalla represents a third mode of regulation, in which an N-terminal catalaselike domain is fused to an 8R-lipoxygenase domain (6). The N-terminal domain (44 kDa) has allene oxide synthase (cAOS) activity with the 8R-hydroperoxide of arachidonic acid (8R- † This research was supported by National Institutes of Health Grant GM36232 (to B.J.G.).* To whom correspondence should be addressed. Phone: (850) 644-8547. Fax: (850) 644-8547. E-mail: gaffney@bio.fsu.edu.. ‡ These authors contributed equally.1 Abbreviations: cAOS, coral allene oxide synthase; BLC, bovine liver catalase; BSA, bovine serum albumin; CYP, cytochrome P450; DES, divinyl ether synthase; EPR, electron paramagnetic resonance; 8R-HpETE, 8R-hydroperoxy-9E,11Z-ei...

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Section: Discussion Stoichiometric Oxidation Of Coral Aos By Peracetimentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Role of Radical Formation at Tyrosine 193 in the Allene Oxide Synthase Domain of a Lipoxygenase−AOS Fusion Protein from Coral

Katsir

Seavy

et al. 2003

Biochemistry

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“…The coral LOX domain has sequence similarities to other mammalian and plant lipoxygenases; the coral AOS domain has significant sequence homology to catalase, albeit with very low (30%) sequence identity (3). It has been shown recently that coral AOS cannot disproportionate hydrogen peroxide like catalases, and catalase cannot convert 8R-HPETE to allene oxide (4).…”

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confidence: 99%

Characterization of the Coral Allene Oxide Synthase Active Site with UV−Visible Absorption, Magnetic Circular Dichroism, and Electron Paramagnetic Resonance Spectroscopy: Evidence for Tyrosinate Ligation to the Ferric Enzyme Heme Iron

et al. 2001

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Coral allene oxide synthase (AOS), a hemoprotein with weak sequence homology to catalase, is the N-terminal domain of a naturally occurring fusion protein with an 8R-lipoxygenase. AOS converts 8R-hydroperoxyeicosatetraenoic acid to the corresponding allene oxide. The UV-visible absorption and magnetic circular dichroism spectra of ferric AOS and of its cyanide and azide complexes, and the electron paramagnetic resonance spectra of native AOS (high-spin, g ) 6.56, 5.22, 2.00) and of its cyanide adduct (low-spin, g ) 2.86, 2.24, 1.60) closely resemble the corresponding spectra of bovine liver catalase (BLC). These results provide strong evidence for tyrosinate ligation to the heme iron of AOS as has been established for catalases. On the other hand, the positive circular dichroism bands in the Soret region for all three derivatives of ferric AOS are almost the mirror image of those in catalase. In addition, the cyanide affinity of native AOS (K d ) 10 mM at pH 7) is about 3 orders of magnitude lower than that of BLC. Thus, while these results conclusively support a common tyrosinate-ligated heme in AOS as in catalase, significant differences exist in the interaction between their respective heme prosthetic groups and protein environments, and in the access of small molecules to the heme iron.Interest in the enzymology underlying the high prostaglandin content of the Caribbean sea whip coral, Plexaura homomalla, has been the impetus for several biochemical investigations. Arachidonic acid is metabolized in extracts of P. homomalla and related corals by a lipoxygenase (LOX) 1 pathway that appeared initially to relate to the production of cyclopentenone prostaglandins (1, 2). An 8R-lipoxygenase converts arachidonic acid to 8R-hydroperoxyeicosatetraenoic acid (8R-HPETE), and the fatty acid hydroperoxide is then enzymatically dehydrated to an allene oxide, an epoxide with a propensity to cyclize to cyclopentenone derivatives. Brash et al., in 1997, isolated the cDNA of an 8R-lipoxygenase from P. homomalla and found it to occur with an extra sequence in the open reading frame that encodes the allene oxide synthase (AOS) (3). This natural fusion protein in coral consists of a LOX C-terminal domain (79 kDa) and an AOS N-terminal domain (43 kDa). The truncated (AOS) construct catalyzes an identical reaction to the AOS domain in the fusion protein, converting 8R-HPETE solely to the allene oxide. The truncated construct exhibits very high turnover number (1400/s) (4), although, due to the weaker expression of the fusion protein in E. coli, the comparable values for the fusion protein remain to be determined. 2 Although allene oxide has not proven to be an intermediate in the prostaglandin synthesis pathway (5), it may be linked to prostanoid type products such as clavulones and punaglandins of other corals (2,6,7). Allene oxides are very unstable epoxides; they readily hydrolyze into ketols, cyclopentenones, and other rearrangement products (8-10).The production of allene oxides from fatty acid hydroperoxides also ...

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The structure and peroxidase activity of a 33‐kDa catalase‐related protein from Mycobacterium avium ssp. paratuberculosis

et al. 2009

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True catalases are tyrosine-liganded, usually tetrameric, hemoproteins with subunit sizes of~55-84 kDa. Recently characterized hemoproteins with a catalase-related structure, yet lacking in catalatic activity, include the 40-43 kDa allene oxide synthases of marine invertebrates and cyanobacteria. Herein, we describe the 1.8 Å X-ray crystal structure of a 33 kDa subunit hemoprotein from Mycobacterium avium ssp. paratuberculosis (annotated as MAP-2744c), that retains the core elements of the catalase fold and exhibits an organic peroxide-dependent peroxidase activity. MAP-2744c exhibits negligible catalatic activity, weak peroxidatic activity using hydrogen peroxide (20/s) and strong peroxidase activity (~300/s) using organic hydroperoxides as co-substrate. Key amino acid differences significantly impact prosthetic group conformation and placement and confer a distinct activity to this prototypical member of a group of conserved bacterial ''minicatalases''. Its structural features and the result of the enzyme assays support a role for MAP-2744c and its close homologues in mitigating challenge by a variety of reactive oxygen species.

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Purification and Catalytic Activities of the Two Domains of the Allene Oxide Synthase-Lipoxygenase Fusion Protein of the CoralPlexaura homomalla

Cited by 70 publications

References 42 publications

Role of Radical Formation at Tyrosine 193 in the Allene Oxide Synthase Domain of a Lipoxygenase−AOS Fusion Protein from Coral

Role of Radical Formation at Tyrosine 193 in the Allene Oxide Synthase Domain of a Lipoxygenase−AOS Fusion Protein from Coral

Characterization of the Coral Allene Oxide Synthase Active Site with UV−Visible Absorption, Magnetic Circular Dichroism, and Electron Paramagnetic Resonance Spectroscopy: Evidence for Tyrosinate Ligation to the Ferric Enzyme Heme Iron

The structure and peroxidase activity of a 33‐kDa catalase‐related protein from Mycobacterium avium ssp. paratuberculosis

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