Alkene Monooxygenase from <i>Nocardia Corallina</i> B‐276 is a Member of the Class of Dinuclear Iron Proteins Capable of Stereospecific Epoxygenation Reactions

Gallagher, Stephen C.; Cammack, Richard; Dalton, Howard

doi:10.1111/j.1432-1033.1997.00635.x

Cited by 70 publications

(90 citation statements)

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“…Solvent-derived water and hydroxide ions complete the octahedral coordination spheres. Sequence, spectroscopic, and structural analyses suggest that all BMM hydroxylases have nearly identical dinuclear iron centers (1,(10)(11)(12). Because AMO, TMO, SsoMO, and PH are incapable of hydroxylating alkanes, whereas sMMO and THFMO are adept at this transformation, other features of the system must control substrate reactivity at their dioxygen-activated metal centers.…”

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confidence: 99%

X-ray Structure of a Hydroxylase−Regulatory Protein Complex from a Hydrocarbon-Oxidizing Multicomponent Monooxygenase, Pseudomonas sp. OX1 Phenol Hydroxylase^,

et al. 2006

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Phenol hydroxylase (PH) belongs to a family of bacterial multicomponent monooxygenases (BMMs) with carboxylate-bridged diiron active sites. Included are toluene/o-xylene (ToMO) and soluble methane (sMMO) monooxygenase. PH hydroxylates aromatic compounds, but unlike sMMO, it cannot oxidize alkanes despite having a similar dinuclear iron active site. Important for activity is formation of a complex between the hydroxylase and a regulatory protein component. To address how structural features of BMM hydroxylases and their component complexes may facilitate the catalytic mechanism and choice of substrate, we determined X-ray structures of native and SeMet forms of the PH hydroxylase (PHH) in complex with its regulatory protein (PHM) to 2.3 Å resolution. PHM binds in a canyon on one side of the (αβγ) 2 PHH dimer, contacting α-subunit helices A, E, and F ∼12 Å above the diiron core. The structure of the dinuclear iron center in PHH resembles that of mixed-valent MMOH, suggesting an Fe(II)Fe(III) oxidation state. Helix E, which comprises part of the iron-coordinating four-helix bundle, has more π-helical character than analogous E helices in MMOH and ToMOH lacking a bound regulatory protein. Consequently, conserved active site Thr and Asn residues translocate to the protein surface, and an ∼6 Å pore opens through the four-helix bundle. Of likely functional significance is a specific hydrogen bond formed between this Asn residue and a conserved Ser side chain on PHM. The PHM protein covers a putative docking site on PHH for the PH reductase, which transfers electrons to the PHH diiron center prior to O 2 activation, † This research was supported by National Institute of General Medical Sciences Grant GM32134 (S.J.L.) and the Italian Ministry of SUPPORTING INFORMATION AVAILABLE BMM hydroxylase and regulatory protein structures and their electrostatic surfaces (Figures S1 and S2), packing of the β-subunit Nterminus and a γ-subunit from an adjacent molecule in the PHH canyon ( Figure S3), comparison of known regulatory protein structures ( Figure S4), sequence alignments of the different regulatory proteins ( Figure S5), models of the PHH diiron center in electron density maps ( Figure S6), comparison of the hydroxylase zinc-binding sequences ( Figure S7), and electron density maps around helix F ( Figure S8). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2007 March 21. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptsuggesting that the regulatory component may function to block undesired reduction of oxygenated intermediates during the catalytic cycle. A series of hydrophobic cavities through the PHH α-subunit, analogous to those in MMOH, may facilitate movement of the substrate to and/or product from the active site pocket. Comparisons between the ToMOH and PHH structures provide insights into their substrate regiospecificities.Bacterial multicomponent monooxygenases...

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confidence: 99%

X-ray Structure of a Hydroxylase−Regulatory Protein Complex from a Hydrocarbon-Oxidizing Multicomponent Monooxygenase, Pseudomonas sp. OX1 Phenol Hydroxylase^,

et al. 2006

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“…as carbon source and grown as described previously uct. It has been shown previously [6] that AMO, on the other [6]. hand, is capable of stereoselective epoxidation of propene to 20-l batch fermentation.…”

Section: Fig 1 Comparison Of the Deduced Amino-acid Sequences Of Thmentioning

confidence: 93%

“…A DEAE-cellubelieve particular residues are able to account for the difference lose (Whatman) FPLC column (250 mmϫ56 mm, 120 ml) in stereoselectivity observed between AMO [6] and soluble equilibrated with buffer B was used for initial seperation of the MMO [7].…”

Section: Fig 1 Comparison Of the Deduced Amino-acid Sequences Of Thmentioning

confidence: 99%

“…components rather than an Ion Exchange D (Whatman) FPLC column as used previously [6]. Purification of the epoxygenase.…”

Section: Fig 1 Comparison Of the Deduced Amino-acid Sequences Of Thmentioning

confidence: 99%

“…It may, therefore, account for the observed difference in stereoselectivity of the epoxygenation reaction catalysed into the epoxygenase active site it appears that the C3 methyl group is clamped in place during the epoxygenation reaction. by AMO [6] and soluble MMO [20].…”

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Sequence‐alignment modelling and molecular docking studies of the epoxygenase component of alkene monooxygenase from Nocardia corallina B‐276

Gallagher

George

Dalton

1998

European Journal of Biochemistry

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Whole cells of Nocardia corallina B-276 catalyse the stereoselective epoxygenation of alkenes to chiral epoxides. The bacterium expresses an enzyme, alkene monooxygenase, which catalyses the epoxygenation reaction stereoselectively. The enzyme consists of a terminal oxygenase (epoxygenase), an NADH-dependent reductase (reductase) and a regulatory component (coupling protein).The epoxygenase component contains a bridged diiron centre similar to that found in the hydroxylase component of soluble methane monooxygenase. Sequence-alignment modelling, supported by chemical modification and fluorescence probing, identified a hydrophobic oxygen/substrate binding site within the epoxygenase. The diiron centre was coordinated by the two His and two Glu residues from two conserved Glu-Xaa-Xaa-His sequences and by two further Glu residues. Molecular docking of substrates and products into the proposed active-site model of the epoxygenase suggested that Ala91 and Ala185 were responsible for the stereoselectivity exerted by AMO. It is proposed that these residues clamped the intermediate and/or product of the reaction, thereby controlling the configuration of the epoxide produced. In soluble methane monooxygenase these residues are replaced by two Gly residues which do not provide sufficient steric hindrance to prevent rotation of the intermediate in the active site and, therefore, the product of the reaction catalysed by this enzyme is achiral.Keywords : monooxygenase ; diiron ; epoxide ; stereoselective ; molecular docking.Prior to the crystallisation of the hydroxylase component of placed in soluble MMO by smaller residues (Cys151, Thr213, Ile217) allowing the binding of methane as well as molecular soluble methane monooxygenase, sequence-alignment modelling was used to predict the three-dimensional structure of the oxygen. This difference demonstrated that, although the two diiron proteins had similar iron coordination chemistry, modificaprotein. In that instance, molecular alignment of the hydroxylase to the three-dimensional structure of the R2 subunit of ribonu-tions in the residues surrounding the diiron cluster could be responsible for the observed differences in catalysis. cleotide reductase was performed in order to predict the structure of the hydroxylase active site [1]. In both proteins the diiron George et al.[4] used molecular dynamics calculations to determine the docking of substrate molecules into the crystal centre is coordinated by two conserved Glu-Xaa-Xaa-His sequences. Despite this similarity, the hydroxylase of soluble structure of the hydroxylase component of soluble MMO to determine the topology of the substrate binding site. By energy methane monooxygenase (MMO) and the R2 subunit of ribonucleotide reductase differ in their function in that R2 can only minimisation of the compounds into a rigid hydroxylase structure at 1500 K, several potential active sites were identified. bind oxygen whereas the hydroxylase can also bind organic subFrom the minimum energy functions for these sites three potenstra...

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Iron Proteins with Dinuclear Active Sites

Kurtz

2005

Encyclopedia of Inorganic Chemistry

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Iron proteins with dinuclear active sites include those containing stable nonheme, non‐sulfur diiron sites. Though still dwarfed by the numbers of heme or iron‐sulfur proteins, the list of proteins known to contain nonheme, non‐sulfur diiron sites has steadily expanded over the past thirty years. These proteins share the following active site properties: (1) two iron atoms separated by 4 Å or less; (2) stable high spin ferrous and ferric oxidation states; (3) terminal histidinyl imidazole, carboxylate and solvent ligands; (4) at least one ligand that bridges the two irons; and (5) magnetic superexchange coupling of the unpaired spins on the two irons mediated by the bridging ligand atom(s). With one possible exception, the bridging ligands are either known or are strongly suspected to include one or two carboxylates. Two distinct subclasses, the purple acid phosphatases and the flavo‐diiron enzymes, seem to have no structural or functional relationships to each other or to any of the other diiron enzymes. The oxygen‐carrying protein, hemerythrin, for many years served as the prototype for nonheme, non‐sulfur diiron proteins. However, its structure and function have turned out to be distinct from those of the O 2 ‐activating enzymes, which now form the largest subclass of these diiron‐containing proteins, with at least seven distinct types known; the most numerous of these are the alkane (including methane) monooxygenases and fatty acyl desaturases. Evidence for catalytically competent high‐valent diiron intermediates resulting from reaction of the diferrous sites with dioxygen have been obtained for at least two of the O 2 ‐activating enzymes. Among the most intriguing and mysterious of the O 2 ‐activating enzymes are the membrane‐bound ‘eight‐His’ diiron desaturases and monooxygenases.

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Alkene Monooxygenase from Nocardia Corallina B‐276 is a Member of the Class of Dinuclear Iron Proteins Capable of Stereospecific Epoxygenation Reactions

Cited by 70 publications

References 32 publications

X-ray Structure of a Hydroxylase−Regulatory Protein Complex from a Hydrocarbon-Oxidizing Multicomponent Monooxygenase, Pseudomonas sp. OX1 Phenol Hydroxylase^,

X-ray Structure of a Hydroxylase−Regulatory Protein Complex from a Hydrocarbon-Oxidizing Multicomponent Monooxygenase, Pseudomonas sp. OX1 Phenol Hydroxylase^,

Sequence‐alignment modelling and molecular docking studies of the epoxygenase component of alkene monooxygenase from Nocardia corallina B‐276

Iron Proteins with Dinuclear Active Sites

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Alkene Monooxygenase from Nocardia Corallina B‐276 is a Member of the Class of Dinuclear Iron Proteins Capable of Stereospecific Epoxygenation Reactions

Cited by 70 publications

References 32 publications

X-ray Structure of a Hydroxylase−Regulatory Protein Complex from a Hydrocarbon-Oxidizing Multicomponent Monooxygenase, Pseudomonas sp. OX1 Phenol Hydroxylase,

X-ray Structure of a Hydroxylase−Regulatory Protein Complex from a Hydrocarbon-Oxidizing Multicomponent Monooxygenase, Pseudomonas sp. OX1 Phenol Hydroxylase,

Sequence‐alignment modelling and molecular docking studies of the epoxygenase component of alkene monooxygenase from Nocardia corallina B‐276

Iron Proteins with Dinuclear Active Sites

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Product

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X-ray Structure of a Hydroxylase−Regulatory Protein Complex from a Hydrocarbon-Oxidizing Multicomponent Monooxygenase, Pseudomonas sp. OX1 Phenol Hydroxylase^,

X-ray Structure of a Hydroxylase−Regulatory Protein Complex from a Hydrocarbon-Oxidizing Multicomponent Monooxygenase, Pseudomonas sp. OX1 Phenol Hydroxylase^,