Crystal Structure of the Toluene/o-Xylene Monooxygenase Hydroxylase from Pseudomonas stutzeri OX1

Sazinsky, Matthew H.; Bard, Joel; Donato, Alberto Di; Lippard, Stephen J.

doi:10.1074/jbc.m400710200

Cited by 188 publications

(186 citation statements)

References 48 publications

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“…BoxB ox-malonate and methane monooxygenase ox (35) (and tolu- ene/o-xylene diiron monooxygenase ox (36)) only reveal minor differences in the ligation pattern. The carboxylate chelated to Fe2 originates from an aspartate in BoxB (Asp-211) and PaaAC (7) and from a glutamate in the other family members.…”

Section: Resultsmentioning

confidence: 99%

“…Although a few diiron center protein-substrate (analog) complexes were structurally characterized (7,36,38), the BoxB: BCA red structure reveals the first one where the substrate is bound to its binding site in a catalytically relevant orientation relative to the diiron center. The shortest distance between the phenyl ring of benzoyl-CoA and the diiron center is 3.5 Å between Fe1 and C2, whereas the corresponding distance to Fe2 is 4.9 Å (Fig.…”

Section: Resultsmentioning

confidence: 99%

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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%

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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“…One begins in the active site pocket and extends to the protein surface close to E214 and E285 (channel 1), which was described previously by Sazinsky et al 9 for the analogous ToMO. The second (channel 2) is a longer passage connecting the active site to the protein surface by passing W167 and exiting close to S395, and is in good agreement with recent experimental data.…”

Section: Discussionmentioning

confidence: 64%

“…In contrast to other cases, this residue is located 30Å away from the active site, near the interface of subunits  and , suggesting that its catalytic influence should be the result of a change in the active site dynamics or in the ligand delivery. Several crystallographic studies of the BMM superfamily, proposed a common channel through the  subunit connecting the diiron center to the surface; this pathway involves surface residues D285 and E214 in T4MO 9,10 . Additional reports described two other hydrophobic cavities, one near the active site pocket and another which is located near the interface of the  and  subunits 1,5,10 .…”

Section: Introductionmentioning

confidence: 99%

Atomic Picture of Ligand Migration in Toluene 4-Monooxygenase

et al. 2014

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Computational modeling combined with mutational and activity assays were used to underline the substrate migration pathways in toluene 4-monooxygenase, a member of the important family of bacterial multicomponent monooxygenases (BMM). In all structurally defined BMM hydroxylases, several hydrophobic cavities in the α-subunit map a preserved path from the protein surface to the diiron active site. Our results confirm the presence of two pathways by which different aromatic molecules can enter/escape the active site. While the substrate is observed to enter from both channels, the more hydrophilic product is withdrawn mainly from the shorter channel ending at residues D285 and E214. The long channel ends in the vicinity of S395, whose variants have been seen to affect activity and specificity. These mutational effects are clearly reproduced and rationalized by the in silico studies. Furthermore, the combined computational and experimental results highlight the importance of residue F269, which is located at the intersection of the two channels.

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“…The specific activity on phenol was 1.2(1) × 10 3 mU/mg of hydroxylase, and the iron content for ToMOH was 4.3(2) as determined by methods reported elsewhere. 24,25 In a typical reaction, a mixture of 1 nmol of ToMOH, 66.57 nmol of ToMOB, 20 nmol of Rieske protein, and 0.1 nmol of ToMOR was diluted to a volume of 2 mL with 20 mM potassium phosphate buffer (pH = 7.4). The mixture was allowed to stand in an ice bath for 10 min.…”

Section: Toluene Monooxygenase-catalyzed Oxidationsmentioning

confidence: 99%

Products from Enzyme-Catalyzed Oxidations of Norcarenes

et al. 2007

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Recent studies revealed that norcarane (bicyclo[4.1.0]heptane) is oxidized to 2-norcarene (bicyclo [4.1.0]-hept-2-ene) and 3-norcarene (bicyclo[4.1.0]hept-3-ene) by iron-containing enzymes and that secondary oxidation products from the norcarenes complicate mechanistic probe studies employing norcarane as the substrate (Newcomb, M.; Chandrasena, R. E. P.; Lansakara-P., D. S. P.; Kim, H.-Y.; Lippard, S. J.; Beauvais, L. G.; Murray, L. J.; Izzo, V.; Hollenberg, P. F.; Coon, M. J., accompanying article). In the present work, the product profiles from oxidations of 2-norcarene and 3-norcarene by several enzymes were determined. Most of the products were identified by GC and GC-mass spectral comparison to authentic samples produced independently; in some cases, stereochemical assignments were made or confirmed by 2-D NMR analysis of the products. The enzymes studied in this work were four cytochrome P450 enzymes, CYP2B1, CYPΔ2E1, CYPΔ2E1 T303A, and CYPΔ2B4, and three diiron-containing enzymes, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), toluene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from Pseudomonas stutzeri OX1. The oxidation products from the norcarenes identified in this work are 2-norcaranone, 3-norcaranone, syn-and anti-2-norcarene oxide, syn-and anti-3-norcarene oxide, syn-and anti-4-hydroxy-2-norcarene, syn-and anti-2-hydroxy-3-norcarene, 2-oxo-3-norcarene, 4-oxo-2-norcarene, and cyclohepta-3,5-dienol. Two additional, unidentified oxidation products were observed in low yields in the oxidations. In matched oxidations, 3-norcarene was a better substrate than 2-norcarene in terms of turnover by factors of 1.5 to 15 for the enzymes studied here. The oxidation products found in enzyme-catalyzed oxidations of the norcarenes are useful for understanding the complex product mixtures obtained in norcarane oxidations.Mechanistic probes have been used for years to reveal details about reaction mechanisms in chemistry and biology. The concept of a mechanistic probe study is that a short-lived intermediate can be revealed by a characteristic rearrangement of a probe substrate that is observed in the reaction products. One compound used as a probe substrate for studies of enzyme-catalyzed oxidations is norcarane, bicyclo[4.1.0]heptane (1). During the course of probe studies with norcarane in our laboratories, 1 we observed a large number of minor oxidation products that appeared to be derived from norcarane as judged by GC retention times and mass spectra. Eventually, we realized that, in addition to expected alcohol products, the enzymes were oxidizing norcarane to both 2-norcarene (2) and 3-norcarene (3), and that these alkenes were further oxidized to give small amounts of secondary oxidation products. of the norcarane oxidation studies are reported in the accompanying paper. 3 In this paper, we report studies of the oxidations of both 2-norcarene and 3-norcarene by several iron-containing enzymes. Most of the products from oxidations of th...

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Crystal Structure of the Toluene/o-Xylene Monooxygenase Hydroxylase from Pseudomonas stutzeri OX1

Cited by 188 publications

References 48 publications

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

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

Atomic Picture of Ligand Migration in Toluene 4-Monooxygenase

Products from Enzyme-Catalyzed Oxidations of Norcarenes

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