Desaturases: Emerging Models for Understanding Functional Diversification of Diiron-containing Enzymes

Shanklin, John; Guy, Jodie E.; Mishra, Girish; Lindqvist, Ylva

doi:10.1074/jbc.r900009200

Cited by 165 publications

(170 citation statements)

References 49 publications

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“…2A). In comparison, other family members such as stearyl-ACP ⌬9 desaturase, ribonucleotide reductase R2, and methane monooxygenase are homodimers, heterotetramers, and heterohexamers, respectively (8,31,32).…”

Section: Resultsmentioning

confidence: 99%

Structure and Mechanism of the Diiron Benzoyl-Coenzyme A Epoxidase BoxB

Rather

Weinert

Demmer

et al. 2011

Journal of Biological Chemistry

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The coenzyme A (CoA)-dependent aerobic benzoate metabolic pathway uses an unprecedented chemical strategy to overcome the high aromatic resonance energy by forming the nonaromatic 2,3-epoxybenzoyl-CoA. The crucial dearomatizing reaction is catalyzed by three enzymes, BoxABC, where BoxA is an NADPH-dependent reductase, BoxB is a benzoyl-CoA 2,3-epoxidase, and BoxC is an epoxide ring hydrolase. We characterized the key enzyme BoxB from Azoarcus evansii by structural and Mössbauer spectroscopic methods as a new member of class I diiron enzymes. Several family members were structurally studied with respect to the diiron center architecture, but no structure of an intact diiron enzyme with its natural substrate has been reported. X-ray structures between 1.9 and 2.5 Å resolution were determined for BoxB in the diferric state and with bound substrate benzoyl-CoA in the reduced state. The substrate-bound reduced state is distinguished from the diferric state by increased iron-ligand distances and the absence of directly bridging groups between them. The position of benzoylCoA inside a 20 Å long channel and the position of the phenyl ring relative to the diiron center are accurately defined. The C2 and C3 atoms of the phenyl ring are closer to one of the irons. Therefore, one oxygen of activated O 2 must be ligated predominantly to this proximate iron to be in a geometrically suitable position to attack the phenyl ring. Consistent with the observed iron/phenyl geometry, BoxB stereoselectively should form the 2S,3R-epoxide. We postulate a reaction cycle that allows a charge delocalization because of the phenyl ring and the electron-withdrawing CoA thioester.The metabolism of aromatic compounds has attracted broad attention due to its importance in the biogeochemical carbon cycle (includes 10 -20% of the biomass) and because of the chemistry necessary to overcome the high resonance energy of the conjugated cyclic ring. A central intermediate of the aromatic metabolism is benzoate, which can be degraded by microorganisms using three different strategies. The first strategy in the presence of oxygen uses mono-and dioxygenases to hydroxylate benzoate to catechol or protocatechuate. Central ring-cleaving dioxygenases with mononuclear iron centers subsequently cleave the aromatic ring either between the two hydroxyl groups (ortho cleavage, ␤-ketoadipate pathway) or next to one of the hydroxyl groups (meta cleavage) (1). The second strategy under anoxic conditions involves benzoylCoA, which is reduced by two electrons to a non-aromatic cyclic diene followed by hydrolytic ring opening. Benzoyl-CoA reduction is accomplished by two completely different enzyme systems that may catalyze a Birch-like reduction (2).The third, semi-aerobic strategy to metabolize benzoate shares characteristic features of both of these options (3). Oxygen is still required for attacking the ring, as in the aerobic metabolism, whereas all metabolites are activated to CoA thioesters and ring cleavage is performed hydrolytically, as in the anaerobic pathwa...

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Section: Resultsmentioning

confidence: 99%

Structure and Mechanism of the Diiron Benzoyl-Coenzyme A Epoxidase BoxB

Rather

Weinert

Demmer

et al. 2011

Journal of Biological Chemistry

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“…Although regiospecific Cβ−H atom abstraction would most certainly need to be primarily satisfied, it does not solely ensure subsequent overoxidation to furnish a hydrocarbon by OleT and related CYP152 orthologs (40,52). Interesting comparisons are found with the functional divergence observed for nonheme iron enzymes and synthetic complexes, which effectively negotiate • OH bond insertion, rebound of alternative radical species (22), and substrate desaturation (50,53). We anticipate that further elucidation of the factors that promote alkene formation will provide important parallels with these systems in a different structural framework, and provide opportunities to better leverage OleT activity with a broader substrate scope.…”

Section: Significancementioning

confidence: 98%

“…We propose, based on analogy to mechanisms purported for desaturases, which can also exhibit dual functionality (reviewed in ref. 50), that bifurcation of the two pathways results from a competition of • OH rebound and abstraction of an additional substrate electron by Ole-II. The latter pathway, coupled to the recruitment of a proton to restore Fe 3+ −OH 2 (Fig.…”

Section: Significancementioning

confidence: 99%

Catalytic strategy for carbon−carbon bond scission by the cytochrome P450 OleT

Grant

Mitchell

Makris

2016

Proc. Natl. Acad. Sci. U.S.A.

102

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OleT is a cytochrome P450 that catalyzes the hydrogen peroxidedependent metabolism of C n chain-length fatty acids to synthesize C n-1 1-alkenes. The decarboxylation reaction provides a route for the production of drop-in hydrocarbon fuels from a renewable and abundant natural resource. This transformation is highly unusual for a P450, which typically uses an Fe 4+ −oxo intermediate known as compound I for the insertion of oxygen into organic substrates. OleT, previously shown to form compound I, catalyzes a different reaction. A large substrate kinetic isotope effect (≥8) for OleT compound I decay confirms that, like monooxygenation, alkene formation is initiated by substrate C−H bond abstraction. Rather than finalizing the reaction through rapid oxygen rebound, alkene synthesis proceeds through the formation of a reaction cycle intermediate with kinetics, optical properties, and reactivity indicative of an Fe 4+ −OH species, compound II. The direct observation of this intermediate, normally fleeting in hydroxylases, provides a rationale for the carbon−carbon scission reaction catalyzed by OleT.C ytochrome P450 (CYP) enzymes catalyze an extraordinary breadth of physiologically important oxidations for xenobiotic detoxification and specialized biosynthetic pathways (1, 2). The metabolic diversity of CYP enzymes originates from a sophisticated interplay of substrate molecular recognition with precise tuning of metal oxygen species formed at the enzyme active site. CYPs use a thiolate-ligated heme iron cofactor to activate molecular oxygen and produce short-lived ferric superoxo (3-5), ferric (hydro)peroxo (6-8), and ferryl (9, 10) intermediates. Coordinated efforts over several decades have resulted in isolation of each intermediate, including recent characterization of the principal oxidant thought to be responsible for the vast majority of P450 oxidations, the Fe 4+ −oxo pi−cation radical species commonly referred to as compound I (or P450-I) (10).The archetypal P450 hydroxylation involves C−H bond abstraction by P450-I and ensuing rapid oxygen insertion through a recombination process termed "oxygen rebound" (11, 12). The hydrogen abstraction/rebound mechanism describes the strategy used for most P450 transformations, and is thought to describe catalysis by numerous metal-dependent oxygenases and synthetic bio-inspired metal−oxo complexes (e.g., refs. 13-17). Despite the ubiquity of this mechanism, in some metalloenzymes, a metal−oxo species is formed that is not ultimately destined for incorporation into a substrate. Some characterized examples include the nonheme dinuclear iron ribonucleotide reductase (RNR R2) (18,19) and mononuclear iron halogenase SyrB2 (20-22). The mechanisms for RNR and SyrB2 highlight the importance of quaternary structural elements, an extensive 35-Å proton-coupled electron transfer pathway in RNR (23), and extremely subtle (subangstrom) substrate positional tuning (SyrB2) (21,22), to enable efficient circumvention from the monooxygenation reaction coordinate.P450-I has also been link...

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“…This includes an investigation of chemoselectivity in oleate 12-hydroxylase, regioselectivity in FAD2-like fungal desaturases, and substrate specificity in a fungal Δ6 desaturase (7,(14)(15)(16)(17). It is interesting to note that all of the relevant mutations which showed important changes in hydroxylase activity cluster near the His boxes in these enzymes.…”

Section: Discussionmentioning

confidence: 99%

Structural Control of Chemoselectivity, Stereoselectivity, and Substrate Specificity in Membrane-Bound Fatty Acid Acetylenases and Desaturases

et al. 2009

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Structural control of chemoselectivity, stereoselectivity, and substrate specificity in membrane-bound fatty acid acetylenases and desaturases Gagné, Steve J.; Reed, Darwin W.; Gray, Gordon R.; Covello, Patrick S. Saskatoon SK S7N 5A8, Canada Received September 14, 2009; Revised Manuscript Received October 26, 2009 ABSTRACT: The FAD2-like desaturases comprise a group of membrane-bound oxygenases involved in the modification of fatty acyl groups in plants and fungi. This group includes typical oleate desaturases which introduce a Δ12 cis double bond and more unusual enzymes such as Crep1, an acetylenase from the plant Crepis alpina, which introduces a triple bond in linoleate at the Δ12 position. In this study, the structure--function relationship between FAD2-like acetylenases and desaturases was examined through site-directed mutagenesis and heterologous expression. Eleven amino acid positions were identified that show complete evolutionary conservation within acetylenases or desaturases but have different amino acids in the other class of enzyme. Point mutants in Crep1 were constructed and expressed in yeast to test the role in fatty acid modification of the amino acids at the 11 positions. Results indicate the importance of five amino acid positions within Crep1 with regard to desaturase and acetylenase chemoselectivity, stereoselectivity, and substrate recognition. For example, relative to wild-type Crep1, the Y150F, F259L, and H266Q mutations all favored desaturation over acetylenation. The data indicate that small changes in primary sequence, particularly in the vicinity of the active site, can have profound changes on chemoselectivity and other aspects of the function of membrane-bound desaturase-like enzymes.The FAD2 1 -like desaturases comprise a group of membranebound oxygenases involved in the modification of fatty acyl groups in plants and fungi. The archetypal plant FAD2 catalyzes the introduction of a cis double bond at the Δ12 position of oleoylphosphatidylcholine in the endoplasmic reticulum (1) (Figure 1). This requires molecular oxygen and reducing equivalents from cytochrome b 5 (2).Interestingly, the FAD2-like enzyme group includes a number of variants which catalyze reactions other than the usual Δ12 cis desaturation. These reactions include hydroxylation, epoxidation, conjugated double bond formation, and triple bond formation (3). The acetylenase Crep1, from Crepis alpina, is the first of a number of FAD2-like acetylenases identified in the plant families Asteraceae, Apiaceae, and Araliaceae (4). Crep1 catalyzes the formation of a triple bond at the 12,13 position of linoleate (18:2-9c,12c) to give crepenynate (18:1-9c,12a) (Figure 1). Crep1 has also been reported to catalyze both cis and trans desaturation of oleate (5) (Figure 1).In general, the membrane-bound desaturases are particularly difficult to work with in vitro.Muchofwhatisknownaboutthe structure and function of these enzymes has been derived from their primary structure and in vivo activities in yeast. By analogy to solub...

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Desaturases: Emerging Models for Understanding Functional Diversification of Diiron-containing Enzymes

Cited by 165 publications

References 49 publications

Structure and Mechanism of the Diiron Benzoyl-Coenzyme A Epoxidase BoxB

Structure and Mechanism of the Diiron Benzoyl-Coenzyme A Epoxidase BoxB

Catalytic strategy for carbon−carbon bond scission by the cytochrome P450 OleT

Structural Control of Chemoselectivity, Stereoselectivity, and Substrate Specificity in Membrane-Bound Fatty Acid Acetylenases and Desaturases

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