Xylene monooxygenase catalyzes the multistep oxygenation of toluene and pseudocumene to corresponding alcohols, aldehydes, and acids in Escherichia coli JM101

Xylene monooxygenase of Pseudomonas putida mt-2 catalyzes the methylgroup hydroxylation of toluene and xylenes. To investigate the potential of xylene monooxygenase to catalyze multistep oxidations of one methyl group, we tested recombinant Escherichia coli expressing the monooxygenase genes xylM and xylA under the control of the alk regulatory system of Pseudomonas oleovorans Gpo1. Expression of xylene monooxygenase genes could efficiently be controlled by n-octane and dicyclopropylketone. Xylene monooxygenase was found to catalyze the oxygenation of toluene, pseudocumene, the corresponding alcohols, and the corresponding aldehydes. For all three transformations (18)O incorporation provided stong evidence for a monooxygenation type of reaction, with gem-diols as the most likely reaction intermediates during the oxygenation of benzyl alcohols to benzaldehydes. To investigate the role of benzyl alcohol dehydrogenase (XylB) in the formation of benzaldehydes, xylB was cloned behind and expressed in concert with xylMA. In comparison to E. coli expressing only xylMA, the presence of xylB lowered product formation rates and resulted in back formation of benzyl alcohol from benzaldehyde. In P. putida mt-2 XylB may prevent the formation of high concentrations of the particularly reactive benzaldehydes. In the case of high fluxes through the degradation pathways and low aldehyde concentrations, XylB may contribute to benzaldehyde formation via the energetically favorable dehydrogenation of benzyl alcohols. The results presented here characterize XylMA as an enzyme able to catalyze the multistep oxygenation of toluenes.     

Characterization and application of xylene monooxygenase for multistep biocatalysis

Xylene monooxygenase of Pseudomonas putida mt-2 catalyzes multistep oxidations of one methyl group of toluene and xylenes. Recombinant Escherichia coli expressing the monooxygenase genes xylM and xylA catalyzes the oxygenation of toluene, pseudocumene, the corresponding alcohols, and the corresponding aldehydes, all by a monooxygenation type of reaction (B. Bühler, A. Schmid, B. Hauer, and B. Witholt, J. Biol. Chem. 275:10085-10092, 2000). Using E. coli expressing xylMA, we investigated the kinetics of this one-enzyme three-step biotransformation. We found that unoxidized substrates like toluene and pseudocumene inhibit the second and third oxygenation steps and that the corresponding alcohols inhibit the third oxygenation step. These inhibitions might promote the energetically more favorable alcohol and aldehyde dehydrogenations in the wild type. Growth of E. coli was strongly affected by low concentrations of pseudocumene and its products. Toxicity and solubility problems were overcome by the use of a two-liquid-phase system with bis(2-ethylhexyl)phthalate as the carrier solvent, allowing high overall substrate and product concentrations. In a fed-batch-based two-liquid-phase process with pseudocumene as the substrate, we observed the consecutive accumulation of aldehyde, acid, and alcohol. Our results indicate that, depending on the reaction conditions, product formation could be directed to one specific product.     

Complete amino acid sequence of ferredoxin from Peridinium bipes (Dinophyceae)

The amino acid sequence of the major ferredoxin component isolated from a dinoflagellate, Peridinium bipes, was completely determined. Staphylococcus aureus V8 proteolytic, tryptic and chymotryptic peptides of Cm-ferredoxin were prepared and sequenced. The sequence was Phe-Lys-Val-Thr-Leu-Asp-Thr-Pro-Asp-Gly-Lys-Lys-Ser-Phe-Glu-Cys- Pro-Gly-Asp-Ser-Tyr-Ile-Leu-Asp-Lys-Ala-Glu-Glu-Glu-Gly-Leu-Glu-Leu-Pro- Tyr-Ser - Cys-Arg-Ala-Gly-Ser-Cys-Ser-Ser-Cys-Ala-Gly-Lys-Val-Leu-Thr-Gly-Ser-Ile- Asp-Gln - Ser-Asp-Gln-Ala-Phe-Leu-Asp-Asp-Asp-Gln-Gly-Gly-Asp-Gly-Tyr-Cys-Leu-Thr- Cys-Val - Thr-Tyr-Pro-Thr-Ser-Asp-Val-Thr-Ile-Lys-Thr-His-Cys-Glu-Ser-Glu-Leu. It was composed of 93 amino acid residues with 7 cysteine residues, the highest number found among the chloroplast-type ferredoxins so far sequenced. A cysteine residue was found for the first time at the 89th position in a chloroplast-type ferredoxin. Calculation of the numbers of amino acid differences among chloroplast-type ferredoxins indicates that the Peridinium ferredoxin is far divergent not only from higher plant ferredoxins but also from blue-green algal ferredoxins.     

Redox and functional analysis of the Rieske ferredoxin component of the toluene 4-monooxygenase

Toluene 4-monooxygenase catalyzes the NADH- and O2-dependent hydroxylation of toluene to form p-cresol. The four-protein complex consists of a diiron hydroxylase, an oxidoreductase, a catalytic effector protein, and a Rieske-type ferredoxin (T4moC). Phylogenetic analysis suggests that T4moC is part of a clade specialized for reaction with diiron hydroxylases, possibly reflected in the conservation of W69, whose indole side chain makes close contacts with a bridging sulfide. In order to further investigate the possible origins of this specialization, T4moC, mutated variants of T4moC, and three other purified ferredoxins (the Thermus Rieske protein, the Burkholderia cepacia Rieske-type biphenyl dioxygenase ferredoxin BphF, and the Ralstonia pickettii PK01 toluene monooxygenase TbuB, the Rieske-type ferredoxin from another diiron monooxygenase complex) were studied by redox potential measurements and their ability to complement the catalytic function of the reconstituted toluene 4-monooxygenase complex. A saturation mutagenesis of T4moC W69 indicates that an aromatic residue may modulate the redox potential and is also necessary for activity and/or stability. The redox potential of T4moC was determined to be -173 mV, W69F T4moC was -139 mV, and TbuB was -150 mV. For comparison, BphF had a redox potential of -157 mV [Couture et al. (2001) Biochemistry 40, 84-92]. Of these ferredoxins, all except BphF were able to provide catalytic activity. Given the range in redox potentials observed in the active ferredoxins, shape and electrostatics are strongly implicated in the catalytic specialization. Mutagenesis of other T4moC surface residues gave further insight into possible origins of catalytic specialization. Thus R65A T4moC gave an alteration in apparent KM only, while D82A/D83A T4moC gave alterations in both apparent kcat and KM. Since the different catalytic results were obtained by mutagenesis of residues lying on different sides of the protein adjacent to the [2Fe-2S] cluster, the results suggest that two different faces of T4moC may be involved in protein-protein interactions during catalysis.     

Nucleotide sequences of the Acinetobacter calcoaceticus benABC genes for benzoate 1,2-dioxygenase reveal evolutionary relationships among multicomponent oxygenases.

The nucleotide sequences of the Acinetobacter calcoaceticus benABC genes encoding a multicomponent oxygenase for the conversion of benzoate to a nonaromatic cis-diol were determined. The enzyme, benzoate 1,2-dioxygenase, is composed of a hydroxylase component, encoded by benAB, and an electron transfer component, encoded by benC. Comparison of the deduced amino acid sequences of BenABC with related sequences, including those for the multicomponent toluate, toluene, benzene, and naphthalene 1,2-dioxygenases, indicated that the similarly sized subunits of the hydroxylase components were derived from a common ancestor. Conserved cysteine and histidine residues may bind a [2Fe-2S] Rieske-type cluster to the alpha-subunits of all the hydroxylases. Conserved histidines and tyrosines may coordinate a mononuclear Fe(II) ion. The less conserved beta-subunits of the hydroxylases may be responsible for determining substrate specificity. Each dioxygenase had either one or two electron transfer proteins. The electron transfer component of benzoate dioxygenase, encoded by benC, and the corresponding protein of the toluate 1,2-dioxygenase, encoded by xylZ, were each found to have an N-terminal region which resembled chloroplast-type ferredoxins and a C-terminal region which resembled several oxidoreductases. These BenC and XylZ proteins had regions similar to certain monooxygenase components but did not appear to be evolutionarily related to the two-protein electron transfer systems of the benzene, toluene, and naphthalene 1,2-dioxygenases. Regions of possible NAD and flavin adenine dinucleotide binding were identified.     

On the specificity of pig adrenal ferredoxin (adrenodoxin) and spinach ferredoxin in electron-transfer reactions

Spinach leaf ferredoxin and ferredoxin:NADP oxidoreductase as well as pig adrenodoxin and adrenodoxin reductase have been purified to homogeneity. Ferredoxin-NADP reductase and adrenodoxin-NADP reductase can perform the same diaphorase reactions (dichloroindophenol, ferricyanide and cytochrome c reduction) albeit not with the same efficiency. Despite the differences in their redox potentials, animal and plant ferredoxins can be used as heterologous substrates by the ferredoxin-NADP reductases from both sources. In heterologous systems, however, the ferredoxin/adrenodoxin concentrations must be increased approximately 100-fold in order to reach rates similar to those obtained in homologous systems. Ferredoxin and adrenodoxin can form complexes with the heterologous reductases as demonstrated by binding experiments on ferredoxin-Sepharose or ferredoxin-NADP-reductase-Sepharose and by the realization of difference spectra. Adrenodoxin also weakly substitutes for ferredoxin in NADP photoreduction, and can be used as an electron carrier in the light activation of the chloroplastic enzyme NADP-dependent malate dehydrogenase. In addition adrenodoxin is a good catalyst of pseudocyclic photophosphorylation, but not of cyclic phosphorylation and can serve as a substrate of glutamate synthase. These results are discussed with respect to the known structures of plant and animals ferredoxins and their respective reductases.     

Examination of protein sequence homologies: V. New perspectives on evolution between bacterial and chloroplast-type ferredoxins inferred from sequence evidence

Summary Sequence homologies among 34 chloroplast-type ferredoxins were examined using a computer program that quantitatively evaluates the extent of sequence similarity as a correlation coefficient. The resultant alignment contains six gaps representing insertions or deletions of some residues, all of which are located such that they precisely preserve the domains of structural fragments as determined by crystallographic data onSpirulina platensis ferredoxin.In the search for any total correlation between the chloroplast-type and 27 bacterial ferredoxins, 1891 comparison matrices prepared for possible combinations indicated that the bacterial basal sequence of 55 residues has been conserved evolutionarily in the chloroplast-type sequences corresponding to residue positions 36–90 ofSpirulina platensis ferredoxin. In addition, the bacterial connector sequence region was found to be conserved. These findings strongly suggest that the bacterial and chloroplast-type ferredoxins descended from a common ancestor, and branched off after the bacterial gene duplication, whereas the chloroplast-type ferredoxins originally were generated by duplicating the already duplicated bacterial gene, i.e., by double-duplication.     

Purification and characterisation of the NADH:acceptor reductase component of xylene monooxygenase encoded by the TOL plasmid pWW0 of Pseudomonas putida mt-2.

The xylene monooxygenase system encoded by the TOL plasmid pWW0 of Pseudomonas putida catalyses the hydroxylation of a methyl side-chain of toluene and xylenes. Genetic studies have suggested that this monooxygenase consists of two different proteins, products of the xylA and xylM genes, which function as an electron-transfer protein and a terminal hydroxylase, respectively. In this study, the electron-transfer component of xylene monooxygenase, the product of xylA, was purified to homogeneity. Fractions containing the xylA gene product were identified by its NADH:cytochrome c reductase activity. The molecular mass of the enzyme was determined to be 40 kDa by SDS/PAGE, and 42 kDa by gel filtration. The enzyme was found to contain 1 mol/mol of tightly but not covalently bound FAD, as well as 2 mol/mol of non-haem iron and 2 mol/mol of acid-labile sulfide, suggesting the presence of two redox centers, one FAD and one [2Fe-2S] cluster/protein molecule. The oxidised form of the protein had absorbance maxima at 457 nm and 390 nm, with shoulders at 350 nm and 550 nm. These absorbance maxima disappeared upon reduction of the protein by NADH or dithionite. The NADH:acceptor reductase was capable of reducing either one- or two-electron acceptors, such as horse heart cytochrome c or 2,6-dichloroindophenol, at an optimal pH of 8.5. The reductase was found to have a Km value for NADH of 22 microM. The oxidation of NADH was determined to be stereospecific; the enzyme is pro-R (class A enzyme). The titration of the reductase with NADH or dithionite yielded three distinct reduced forms of the enzyme: the reduction of the [2Fe-2S] center occurred with a midpoint redox potential of -171 mV; and the reduction of FAD to FAD. (semiquinone form), with a calculated midpoint redox potential of -244 mV. The reduction of FAD. to FAD.. (dihydroquinone form), the last stage of the titration, occurred with a midpoint redox potential of -297 mV. The [2Fe-2S] center could be removed from the protein by treatment with an excess of mersalyl acid. The [2Fe-2S]-depleted protein was still reduced by NADH, giving rise to the formation of the anionic flavin semiquinone observed in the native enzyme, thus suggesting that the electron flow was NADH --> FAD --> [2Fe-2S] in this reductase. The resulting protein could no longer reduce cytochrome c, but could reduce 2,6-dichloroindophenol at a reduced rate.     

Divergent evolution of chloroplast-type ferredoxins

The TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes required for the oxidation of toluene to Krebs cycle intermediates. The structural genes for these enzymes are encoded in two operons which comprise the xylCMABN and xylXYZLTEGFJQKIH genes, respectively. The function of the xylT gene has not yet been identified. The nucleotide sequence of xylT was determined in this study and putative gene product was shown to contain a sequence characteristic for chloroplast-type ferredoxins. The nahT gene, the homologue of xylT, present on NAH plasmid NAH7 encoding naphthalene-degrading enzymes, was also sequenced. The sequence conservation between xylT and nahT strongly suggests that both gene products have some physiological function. Chloroplast-type ferredoxins have been discovered in photosynthetic organisms (plants, algae, cyanobacteria and Rhodobacter) and Halobacterium species. Furthermore, chloroplast-type ferredoxin-like sequences have been found in the electron-transfer components of some oxygenases. The sequences of XylT and NahT were compared with those of the previously identified chloroplast-type ferredoxins, in order to examine their evolutionary relationships.     

Sequence analysis of three plasmids harboured in Rhodococcus erythropolis strain PR4

Rhodococcus erythropolis strain PR4 has been isolated as an alkane-degrading bacterium. The strain harbours one linear plasmid, pREL1 (271 577 bp) and two circular plasmids, pREC1 (104 014 bp) and pREC2 (3637 bp), all with some sequence similarities to other Rhodococcus plasmids. For pREL1, pREC1 and pREC2, 298, 102 and 3 open reading frames, respectively, were predicted. Linear plasmid pREL1 has several regions homologous to plasmid pBD2 found in R. erythropolis BD2. Sequence analysis of pREL1 and pBD2 identified common metal-resistance genes on both, but pREL1 also encodes alkane-degradation genes not found on pBD2, with enzyme constituents some of which are quite different from those of other organisms. The alkane hydroxylase consisted of a cytochrome P450 monooxygenase, a 2Fe-2S ferredoxin, and a ferredoxin reductase. The ferredoxin reductase amino acid sequence resembles the AlkT (rubredoxin reductase) sequence. A zinc-containing alcohol dehydrogenase further oxydizes alkanols, alkane oxidation products catalysed by alkane hydroxylase. Of the circular plasmids, the pREC1 sequence is partially similar to the sequence of pREAT701, the virulence plasmid found in Rhodococcus equi. pREC1 has no pREAT701 virulence genes and encodes genes for beta-oxidation of fatty acids. Thus, joint actions of enzymes encoded by pREL1 and pREC1 may enable efficient mineralization of alkanes.     

NMR study of the electron transfer complex of plant ferredoxin and sulfite reductase: mapping the interaction sites of ferredoxin

Plant ferredoxin serves as the physiological electron donor for sulfite reductase, which catalyzes the reduction of sulfite to sulfide. Ferredoxin and sulfite reductase form an electrostatically stabilized 1:1 complex for the intermolecular electron transfer. The protein-protein interaction between these proteins from maize leaves was analyzed by nuclear magnetic resonance spectroscopy. Chemical shift perturbation and cross-saturation experiments successfully mapped the location of two major interaction sites of ferredoxin: region 1 including Glu-29, Glu-30, and Asp-34 and region 2 including Glu-92, Glu-93, and Glu-94. The importance of these two acidic patches for interaction with sulfite reductase was confirmed by site-specific mutation of acidic ferredoxin residues in regions 1 and 2, separately and in combination, by which the ability of mutant ferredoxins to transfer electrons and bind to sulfite reductase was additively lowered. Taken together, this study gives a clear illustration of the molecular interaction between ferredoxin and sulfite reductase. We also present data showing that this interaction surface of ferredoxin significantly differs from that when ferredoxin-NADP(+) reductase is the interaction partner.     

NMR structure of the flavin domain from soluble methane monooxygenase reductase from Methylococcus capsulatus (Bath)

Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane by dioxygen to methanol, the first step in carbon assimilation by methanotrophs. This multicomponent system transfers electrons from NADH through a reductase component to the non-heme diiron center in the hydroxylase where O(2) is activated. The reductase component comprises three distinct domains, a [2Fe-2S] ferredoxin domain along with FAD- and NADH-binding domains. We report the solution structure of the reduced 27.6 kDa FAD- and NADH-binding domains (MMOR-FAD) of the reductase from Methylococcus capsulatus (Bath). The FAD-binding domain consists of a six-stranded antiparallel beta-barrel and one alpha-helix, with the first 10 N-terminal residues unstructured. In the interface between the two domains, the FAD cofactor is tightly bound in an unprecedented extended conformation. The NADH-binding domain consists of a five-stranded parallel beta-sheet with four alpha-helices packing closely around this sheet. MMOR-FAD is structurally homologous to other FAD-containing oxidoreductases, and we expect similar structures for the FAD/NADH-binding domains of reductases that occur in other multicomponent monooxygenases.     

Biochemical and crystallographic characterization of ferredoxin-NADP(+) reductase from nonphotosynthetic tissues.

Distinct forms of ferredoxin-NADP(+) reductase are expressed in photosynthetic and nonphotosynthetic plant tissues. Both enzymes catalyze electron transfer between NADP(H) and ferredoxin; whereas in leaves the enzyme transfers reducing equivalents from photoreduced ferredoxin to NADP(+) in photosynthesis, in roots it has the opposite physiological role, reducing ferredoxin at the expense of NADPH mainly for use in nitrate assimilation. Here, structural and kinetic properties of a nonphotosynthetic isoform were analyzed to define characteristics that may be related to tissue-specific function. Compared with spinach leaf ferredoxin-NADP(+) reductase, the recombinant corn root isoform showed a slightly altered absorption spectrum, a higher pI, a >30-fold higher affinity for NADP(+), greater susceptibility to limited proteolysis, and an approximately 20 mV more positive redox potential. The 1.7 A resolution crystal structure is very similar to the structures of ferredoxin-NADP(+) reductases from photosynthetic tissues. Four distinct structural features of this root ferredoxin-NADP(+) reductases are an alternate conformation of the bound FAD molecule, an alternate path for the amino-terminal extension, a disulfide bond in the FAD-binding domain, and changes in the surface that binds ferredoxin.     

Assessment of the Microbiological Potential for the Natural Attenuation of Petroleum Hydrocarbons in a Shallow Aquifer System

Abstract A multidisciplinary field study investigating the fate and transport of petroleum hydrocarbons commonly associated with jet-fuel contamination is currently underway at Columbus Air Force Base (AFB), Mississippi. Sixty sediment cores from 12 boreholes were recovered from the study aquifer. The goal of this initial sampling was to characterize the potential microbial activity using 14C-labeled substrates, as well as the presence, abundance, and distribution of specific hydrocarbon degrading genotypes using DNA:DNA hybridization. Enumeration of total microbial abundance using a 16S rDNA universal oligonucleotide probe was compared to traditional enumeration methods. Total culturable populations determined by spread plate analysis ranged from a low of 10(4) to more than 10(6) organisms per gram sediment. Microbial abundance estimated by DNA hybridization studies with 16S rDNA genes ranged from 10(7) to 10(8) organisms per gram sediment. Molecular analysis of aquifer samples using DNA probes targeting genes encoding the degradative enzymes alkane hydroxylase (alkB), catechol 2,3-dioxygenase (nahH), naphthalene dioxygenase (nahA), toluene dioxygenase (todC1C2), toluene monooxygenase (tomA), and xylene monooxygenase (xylA), as well as two probes measuring methanogenic microorganisms, codh (carbon monoxide dehydrogenase) and mcr (methyl coenzyme reductase), revealed that each target gene sequence was present in nearly all 60 samples. The presence of organisms demonstrating the phenotype to degrade BTEX and naphthalene was further supported using mineralization assays with 14C-labeled benzene, toluene, naphthalene, and phenanthrene. Minimal activity occurred during the first 24 hours. After a period of 5-7 days, greater than 40% of the target compounds were mineralized in aquifer sediments.     

Solution structure of the toluene 4-monooxygenase effector protein (T4moD)

Toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina catalyzes the NADH- and O(2)-dependent hydroxylation of toluene to form p-cresol. The complex consists of an NADH oxidoreductase (T4moF), a Rieske ferredoxin (T4moC), a diiron hydroxylase [T4moH, with (alphabetagamma)(2) quaternary structure], and a catalytic effector protein (T4moD). The solution structure of the 102-amino acid T4moD effector protein has been determined from 2D and 3D (1)H, (13)C, and (15)N NMR spectroscopic data. The structural model was refined through simulated annealing by molecular dynamics in torsion angle space (DYANA software) with input from 1467 experimental constraints, comprising 1259 distance constraints obtained from NOEs, 128 dihedral angle constraints from J-couplings, and 80 hydrogen bond constraints. Of 60 conformers that met the acceptance criteria, the 20 that best satisfied the input constraints were selected to represent the solution structure. With exclusion of the ill-defined N- and C-terminal segments (Ser1-Asn11 and Asp99-Met102), the atomic root-mean-square deviation for the 20 conformers with respect to the mean coordinates was 0.71 A for the backbone and 1.24 A for all non-hydrogen atoms. The secondary structure of T4moD consists of three alpha-helices and seven beta-strands arranged in an N-terminal betaalphabetabeta and a C-terminal betaalphaalphabetabetabeta domain topology. Although the published NMR structures of the methane monooxygenase effector proteins from Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath) have a similar secondary structure topology, their three-dimensional structures differ from that of T4moD. The major differences in the structures of the three effector proteins are in the relative orientations of the two beta-sheets and the interactions between the alpha-helices in the two domains. The structure of T4moD is closer to that of the methane monooxygenase effector protein from M. capsulatus (Bath) than that from M. trichosporium OB3b. The specificity of T4moD as an effector protein was investigated by replacing it in reconstituted T4MO complexes with effector proteins from monooxygenases from other bacterial species: Pseudomonas pickettii PKO1 (TbuV, toluene 3-monooxygenase); Pseudomonas species JS150 (TbmC, toluene 2-monooxygenase); and Burkeholderia cepacia G4 (S1, toluene 2-monooxygenase). The results showed that the closely related TbuV effector protein (55% sequence identity) provided partial activation of the complex, whereas the more distantly related TbmC (34% sequence identity) and S1 (29% sequence identity) did not. The (1)H NMR chemical shifts of the side-chain amide protons of Asn34, a conserved, structurally relevant amino acid, were found to be similar in spectra of effector proteins T4moD and TbuV but not in the spectrum of TbmC. This suggests that the region around Asn34 may be involved in structural aspects contributing to functional specificity.     

Molecular analysis of a novel methanesulfonic acid monooxygenase from the methylotroph Methylosulfonomonas methylovora

Methylosulfonomonas methylovora M2 is an unusual gram-negative methylotrophic bacterium that can grow on methanesulfonic acid (MSA) as the sole source of carbon and energy. Oxidation of MSA by this bacterium is carried out by a multicomponent MSA monooxygenase (MSAMO). Cloning and sequencing of a 7.5-kbp SphI fragment of chromosomal DNA revealed four tightly linked genes encoding this novel monooxygenase. Analysis of the deduced MSAMO polypeptide sequences indicated that the enzyme contains a two-component hydroxylase of the mononuclear-iron-center type. The large subunit of the hydroxylase, MsmA (48 kDa), contains a typical Rieske-type [2Fe-2S] center with an unusual iron-binding motif and, together with the small subunit of the hydroxylase, MsmB (20 kDa), showed a high degree of identity with a number of dioxygenase enzymes. However, the other components of the MSAMO, MsmC, the ferredoxin component, and MsmD, the reductase, more closely resemble those found in other classes of oxygenases. MsmC has a high degree of identity to ferredoxins from toluene and methane monooxygenases, which are enzymes characterized by possessing hydroxylases containing mu-oxo bridge binuclear iron centers. MsmD is a reductase of 38 kDa with a typical chloroplast-like [2Fe-2S] center and conserved flavin adenine dinucleotide- and NAD-binding motifs and is similar to a number of mono- and dioxygenase reductase components. Preliminary analysis of the genes encoding MSAMO from a marine MSA-degrading bacterium, Marinosulfonomonas methylotropha, revealed the presence of msm genes highly related to those found in Methylosulfonomonas, suggesting that MSAMO is a novel type of oxygenase that may be conserved in all MSA-utilizing bacteria.     

Genetic analysis of functional differences among distinct ferredoxins in Rhodobacter capsulatus

Rhodobacter capsulatus has been known to possess two ferredoxins (I and II) with distinct physicochemical and structural properties: ferredoxin I is a 2[4Fe-4S] type and the other is a [3Fe-4S] [4Fe-4S] type. To analyze their possible functional differences, their genes (fdxN and fdxA) were cloned, sequenced, and subjected to interposon mutagenesis experiments. The former gene was adjacent to a gene encoding a chloroplast-type [2Fe-2S] ferredoxin (fdxC). Mutants with inactivated fdxN and/or fdxC were obtained, and they showed virtually no growth under nitrogen-fixing conditions. Complementation experiments confirmed that both fdxN and fdxC were required for nitrogen fixation. On the other hand, we have not been able to disrupt fdxA under the screening conditions surveyed, including conditions that do not require nitrogenase activity for growth, suggesting that ferredoxin II could have an unknown essential role(s). These indicate functional differences among multiple ferredoxins in one bacterium other than in cyanobacterial heterocysts and indispensability of certain ferredoxins in nitrogen fixation other than Rhizobium meliloti FdxN.     

Amino acid sequence of a four-iron-four-sulphur ferredoxin isolated from Bacillus stearothermophilus.

1. The primary structure of a 4Fe-4S ferredoxin from Bacillus stearothermophilus was determined and shown to consist of a single polypeptide chain of 81 amino acid residues. The molecular weight of the holoprotein is about 9120. 2. There are only four cysteine residues in the molecule; three of these are located near the N-terminus as a Cys-X-X-Cys-X-X-Cys segment, and the fourth cysteine residue is followed by a proline and located in the C-terminal half. 3. The Fe-S chromophore in B. stearothermophilus ferredoxin was previously well characterized and was shown to consist of a single 4Fe-4S cluster. This ferredoxin sequence establishes for the first time the relative location of the four cysteine residues necessary to bind the 4Fe-4S cluster of a 4Fe ferredoxin, and is in agreement with the criteria for the relative positions of the cysteines proposed from X-ray-crystallographic studies on an 8Fe (two 4Fe-4S clusters) ferredoxin. 4. The sequence of B. stearothermophilus ferredoxin is homologous in many segments to that of other bacterial ferredoxins, the degree of homology being greater towards ferredoxins from Desulfovibrio gigas and photosynthetic bacteria than to Clostridial ferredoxins. 5. The presence of a relatively higher number of glutamic acid and lower number of cysteine residues in the molecule may explain the greater thermal stability and oxygen-insenstivity of this ferredoxin.     

Two-step purification of Cyanophora ferredoxin and its identification in soluble protein preparations by isoelectric focusing.

Cyanophora paradoxa ferredoxin is encoded by (cyano-)plastidic DNA, in contrast to those of all other photosynthetic eukaryotes investigated so far. In the present study we report (i) the rapid purification of a chloroplast-type [2Fe-2S] ferredoxin in a two-step procedure by DEAE-Sephadex and Mono Q ion-exchange chromatography; (ii) the biochemical characterization of the purified ferredoxin by electrophoretic separation methods on a microscale; and (iii) a qualitative and quantitative ferredoxin detection method in the femtomole range that allows densitometry, semidry immunoblotting, identification of ferredoxin in soluble cell protein preparations, and analysis of protein biosynthesis from cyanoplast poly(A)- RNA in vivo and in vitro. These fast micromethods should be useful for screening phototrophic species containing ferredoxins encoded by nonnuclear DNA.