Blue protein with red fluorescence.

The walleye (Sander vitreus) is a golden yellow fish that inhabits the Northern American lakes. The recent sightings of the blue walleye and the correlation of its sighting to possible increased UV radiation have been proposed earlier. The underlying molecular basis of its adaptation to increased UV radiation is the presence of a protein (Sandercyanin)-ligand complex in the mucus of walleyes. Degradation of heme by UV radiation results in the formation of Biliverdin IXα (BLA), the chromophore bound to Sandercyanin. We show that Sandercyanin is a monomeric protein that forms stable homotetramers on addition of BLA to the protein. A structure of the Sandercyanin-BLA complex, purified from the fish mucus, reveals a glycosylated protein with a lipocalin fold. This protein-ligand complex absorbs light in the UV region (λmax of 375 nm) and upon excitation at this wavelength emits in the red region (λmax of 675 nm). Unlike all other known biliverdin-bound fluorescent proteins, the chromophore is noncovalently bound to the protein. We provide here a molecular rationale for the observed spectral properties of Sandercyanin.

Structures of the multicomponent Rieske non-heme iron toluene 2,3-dioxygenase enzyme system.

Bacterial Rieske non-heme iron oxygenases catalyze the initial hydroxylation of aromatic hydrocarbon substrates. The structures of all three components of one such system, the toluene 2,3-dioxygenase system, have now been determined. This system consists of a reductase, a ferredoxin and a terminal dioxygenase. The dioxygenase, which was cocrystallized with toluene, is a heterohexamer containing a catalytic and a structural subunit. The catalytic subunit contains a Rieske [2Fe-2S] cluster and mononuclear iron at the active site. This iron is not strongly bound and is easily removed during enzyme purification. The structures of the enzyme with and without mononuclear iron demonstrate that part of the structure is flexible in the absence of iron. The orientation of the toluene substrate in the active site is consistent with the regiospecificity of oxygen incorporation seen in the product formed. The ferredoxin is Rieske type and contains a [2Fe-2S] cluster close to the protein surface. The reductase belongs to the glutathione reductase family of flavoenzymes and consists of three domains: an FAD-binding domain, an NADH-binding domain and a C-terminal domain. A model for electron transfer from NADH via FAD in the reductase and the ferredoxin to the terminal active-site mononuclear iron of the dioxygenase is proposed.

Structural investigations of the ferredoxin and terminal oxygenase components of the biphenyl 2,3-dioxygenase from Sphingobium yanoikuyae B1.

BACKGROUND: The initial step involved in oxidative hydroxylation of monoaromatic and polyaromatic compounds by the microorganism Sphingobium yanoikuyae strain B1 (B1), previously known as Sphingomonas yanoikuyae strain B1 and Beijerinckia sp. strain B1, is performed by a set of multiple terminal Rieske non-heme iron oxygenases. These enzymes share a single electron donor system consisting of a reductase and a ferredoxin (BPDO-FB1). One of the terminal Rieske oxygenases, biphenyl 2,3-dioxygenase (BPDO-OB1), is responsible for B1's ability to dihydroxylate large aromatic compounds, such as chrysene and benzo[a]pyrene. RESULTS: In this study, crystal structures of BPDO-OB1 in both native and biphenyl bound forms are described. Sequence and structural comparisons to other Rieske oxygenases show this enzyme to be most similar, with 43.5 % sequence identity, to naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. While structurally similar to naphthalene 1,2-dioxygenase, the active site entrance is significantly larger than the entrance for naphthalene 1,2-dioxygenase. Differences in active site residues also allow the binding of large aromatic substrates. There are no major structural changes observed upon binding of the substrate. BPDO-FB1 has large sequence identity to other bacterial Rieske ferredoxins whose structures are known and demonstrates a high structural homology; however, differences in side chain composition and conformation around the Rieske cluster binding site are noted. CONCLUSION: This is the first structure of a Rieske oxygenase that oxidizes substrates with five aromatic rings to be reported. This ability to catalyze the oxidation of larger substrates is a result of both a larger entrance to the active site as well as the ability of the active site to accommodate larger substrates. While the biphenyl ferredoxin is structurally similar to other Rieske ferredoxins, there are distinct changes in the amino acids near the iron-sulfur cluster. Because this ferredoxin is used by multiple oxygenases present in the B1 organism, this ferredoxin-oxygenase system provides the structural platform to dissect the balance between promiscuity and selectivity in protein-protein electron transport systems.

NO binding to naphthalene dioxygenase.

Nitric oxide (NO) is commonly used as an analogue for dioxygen in structural and spectroscopic studies of oxygen binding and oxygen activation. In this study, crystallographic structures of naphthalene dioxygenase (NDO) in complex with nitric oxide are reported. In the presence of the aromatic substrate indole, NO is bound end-on to the active-site mononuclear iron of NDO. The structural observations correlate well with spectroscopic measurements of NO binding to NDO in solution. However, the end-on binding of NO is in contrast to the recently reported structure of oxygen to the active-site iron of NDO that binds side-on. While NO is a good oxygen analogue with many similarities to O(2), the different binding mode of NO to the active-site iron atom leads to different mechanistic implications. Hence, caution needs to be used in extrapolating NO as an analogue to O(2) binding.

Structural insight into the dioxygenation of nitroarene compounds: the crystal structure of nitrobenzene dioxygenase.

Nitroaromatic compounds are used extensively in many industrial processes and have been released into the environment where they are considered environmental pollutants. Nitroaromatic compounds, in general, are resistant to oxidative attack due to the electron-withdrawing nature of the nitro groups and the stability of the benzene ring. However, the bacterium Comamonas sp. strain JS765 can grow with nitrobenzene as a sole source of carbon, nitrogen and energy. Biodegradation is initiated by the nitrobenzene dioxygenase (NBDO) system. We have determined the structure of NBDO, which has a hetero-hexameric structure similar to that of several other Rieske non-heme iron dioxygenases. The catalytic subunit contains a Rieske iron-sulfur center and an active-site mononuclear iron atom. The structures of complexes with substrates nitrobenzene and 3-nitrotoluene reveal the structural basis for its activity with nitroarenes. The substrate pocket contains an asparagine residue that forms a hydrogen bond to the nitro-group of the substrate, and orients the substrate in relation to the active-site mononuclear iron atom, positioning the molecule for oxidation at the nitro-substituted carbon.

Purification, crystallization and preliminary X-ray diffraction studies of the three components of the toluene 2,3-dioxygenase enzyme system.

Pseudomonas putida F1 can grow with toluene as its sole source of carbon and energy. The initial reaction of the degradation of toluene is catalyzed by a three-component toluene dioxygenase enzyme system consisting of a reductase (ReductaseTOL), a ferredoxin (FerredoxinTOL) and a Rieske non-heme iron dioxygenase (OxygenaseTOL). The three components and the apoenzyme of the dioxygenase (apo-OxygenaseTOL) were overexpressed, purified and crystallized. ReductaseTOL diffracts to 1.8 A and belongs to space group P4(1)2(1)2, with unit-cell parameters a = b = 77.1, c = 156.3 A. Ferredoxin(TOL) diffracts to 1.2 A and belongs to space group P2(1), with unit-cell parameters a = 30.5, b = 52.0, c = 30.95 A, beta = 113.7 degrees. Apo-OxygenaseTOL and OxygenaseTOL diffract to 3.2 A and belong to space group P4(3)32, with unit-cell parameters a = 235.9 A and a = 234.5 A, respectively.

Stereochemical and mechanistic aspects of dioxygenase-catalysed benzylic hydroxylation of indene and chromane substrates.

Toluene dioxygenase (TDO)-catalysed benzylic hydroxylation of indene substrates (8, 16 and 17), using whole cell cultures of Pseudomonas putida UV4, was found to yield inden-1-ol (14 and 22) and indan-1-one bioproducts (15 and 23). The formation of these bioproducts is consistent with the involvement of carbon-centred radical intermediates. TDO-catalysed oxidation of indenes 8 and 16 also gave cis-diols 13 and 18 respectively. TDO and naphthalene dioxygenase (NDO), used as both whole-cell preparations and as purified enzymes, were found to catalyse the benzylic hydroxylation of chromane 30, deuteriated (+/-)-chromane 30D and enantiomers (4S)-30D and (4R)-30D to yield (4R)- and (4S)-chroman-4-ols 31/31D respectively. The mechanism of benzylic hydroxylation of chromane 30/30D involves the stereoselective abstraction of a pro-R (with TDO) or a pro-S (with NDO) hydrogen atom at C-4 and a marked preference for retention of configuration.

Expression of the nitroarene dioxygenase genes in Comamonas sp. strain JS765 and Acidovorax sp. strain JS42 is induced by multiple aromatic compounds.

This work reports a genetic analysis of the expression of nitrobenzene dioxygenase (NBDO) in Comamonas sp. strain JS765 and 2-nitrotoluene dioxygenase (2NTDO) in Acidovorax sp. strain JS42. Strains JS765 and JS42 possess identical LysR-type regulatory proteins, NbzR and NtdR, respectively. NbzR/NtdR is homologous to NahR, the positive salicylate-responsive transcriptional activator of the naphthalene degradation genes in Pseudomonas putida G7. The genes encoding NBDO and 2NTDO in each strain are cotranscribed, and transcription starts at the same site within identical promoter regions for each operon. Results from a lacZ reporter gene fusion demonstrated that expression of NBDO and 2NTDO is induced by multiple aromatic compounds, including an array of nitroaromatic compounds (nitrobenzene, 2-, 3-, and 4-nitrotoluene, 2,4- and 2,6-dinitrotoluene, and aminodinitrotoluenes), as well as salicylate and anthranilate. The nitroaromatic compounds appear to be the actual effector molecules. Analysis of beta-galactosidase and 2NTDO activities with strain JS42 demonstrated that NtdR was required for induction by all of the inducing compounds, high basal-level expression of 2NTDO, and complementation of a JS42 ntdR null mutant. Complementation with the closely related regulators NagR (from Ralstonia sp. strain U2) and NahR restored only induction by the archetype inducers, salicylate or salicylate and anthranilate, respectively, and did not restore the high basal level of expression of 2NTDO. The mechanism of 2NTDO gene regulation in JS42, and presumably that of NBDO gene regulation in JS765, appear similar to that of NahR-regulated genes in Pseudomonas putida G7. However, NbzR and NtdR appear to have evolved a broader specificity in JS42 and JS765, allowing for recognition of nitroaromatic compounds while retaining the ability to respond to salicylate and anthranilate. NtdR is also the first example of a nitroarene-responsive LysR-type transcriptional activator.

Crystal structure of naphthalene dioxygenase: side-on binding of dioxygen to iron.

Binding of oxygen to iron is exploited in several biological and chemical processes. Although computational and spectroscopic results have suggested side-on binding, only end-on binding of oxygen to iron has been observed in crystal structures. We have determined structures of naphthalene dioxygenase that show a molecular oxygen species bound to the mononuclear iron in a side-on fashion. In a complex with substrate and dioxygen, the dioxygen molecule is lined up for an attack on the double bond of the aromatic substrate. The structures reported here provide the basis for a reaction mechanism and for the high stereospecificity of the reaction catalyzed by naphthalene dioxygenase.

Molecular characterization and substrate specificity of nitrobenzene dioxygenase from Comamonas sp. strain JS765.

Comamonas sp. strain JS765 can grow with nitrobenzene as the sole source of carbon, nitrogen, and energy. We report here the sequence of the genes encoding nitrobenzene dioxygenase (NBDO), which catalyzes the first step in the degradation of nitrobenzene by strain JS765. The components of NBDO were designated Reductase(NBZ), Ferredoxin(NBZ), Oxygenase(NBZalpha), and Oxygenase(NBZbeta), with the gene designations nbzAa, nbzAb, nbzAc, and nbzAd, respectively. Sequence analysis showed that the components of NBDO have a high level of homology with the naphthalene family of Rieske nonheme iron oxygenases, in particular, 2-nitrotoluene dioxygenase from Pseudomonas sp. strain JS42. The enzyme oxidizes a wide range of substrates, and relative reaction rates with partially purified Oxygenase(NBZ) revealed a preference for 3-nitrotoluene, which was shown to be a growth substrate for JS765. NBDO is the first member of the naphthalene family of Rieske nonheme iron oxygenases reported to oxidize all of the isomers of mono- and dinitrotoluenes with the concomitant release of nitrite.