Cloning, heterologous expression, and functional characterization of the nicotinate dehydrogenase gene from Pseudomonas putida KT2440

6-Hydroxynicotinate can be used for the production of drugs, pesticides and intermediate chemicals. Some Pseudomonas species were reported to be able to convert nicotinic acid to 6-hydroxynicotinate by nicotinate dehydrogenase. So far, previous reports on NaDH in Pseudomonas genus were confused and contradictory each other. Recently, Ashraf et al. reported an NaDH gene cloned from Eubacterium barkeri and suggested some deducted NaDH genes from other nine bacteria. But they did not demonstrate the activity of recombinant NaDH and did not mention NaDH gene in Pseudomonas. In this study we cloned the gene of NaDH, ndhSL, from Pseudomonas putida KT2440. NdhSL in P. putida KT2440 is composed of two subunits. The small subunit contains [2Fe2S] iron sulfur domain, while the large subunit contains domains of molybdenum cofactor and cytochrome c. Expression of recombinant ndhSL in P. entomophila L48, which lacks the ability to produce 6-hydroxynicotinate, enabled the resting cell and cell extract of engineering P. entomophila L48 to hydroxylate nicotinate. Gene knockout and recovery studies further confirmed the ndhSL function.     

Molybdenum-Containing Nicotine Hydroxylase Genes in a Nicotine Degradation Pathway That Is a Variant of the Pyridine and Pyrrolidine Pathways

Ochrobactrum sp. strain SJY1 utilizes nicotine as a sole source of carbon, nitrogen, and energy via a variant of the pyridine and pyrrolidine pathways (the VPP pathway). Several strains and genes involved in the VPP pathway have recently been reported; however, the first catalyzing step for enzymatic turnover of nicotine is still unclear. In this study, a nicotine hydroxylase for the initial hydroxylation step of nicotine degradation was identified and characterized. The nicotine hydroxylase (VppA), which converts nicotine to 6-hydroxynicotine in the strain SJY1, is encoded by two open reading frames (vppA_S and vppA_L [subunits S and L, respectively]). The vppA genes were heterologously expressed in the non-nicotine-degrading strains Escherichia coli DH5α and Pseudomonas putida KT2440; only the Pseudomonas strain acquired the ability to degrade nicotine. The small subunit of VppA contained a [2Fe-2S] cluster-binding domain, and the large subunit of VppA contained a molybdenum cofactor-binding domain; however, an FAD-binding domain was not found in VppA. Resting cells cultivated in a molybdenum-deficient medium had low nicotine transformation activity, and excess molybdenum was detected in the purified VppA by inductively coupled plasma-mass spectrometry analysis. Thus, it is demonstrated that VppA is a two-component molybdenum-containing hydroxylase.     

Cloning, nucleotide sequence, and expression of the plasmid-encoded genes for the two-component 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS.

The two-component nonheme iron dioxygenase system 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS catalyzes the double hydroxylation of 2-halobenzoates with concomitant release of halogenide and carbon dioxide, yielding catechol. The gene cluster encoding this enzyme, cbdABC, was localized on a 70-kbp conjugative plasmid designated pBAH1. The nucleotide sequences of cbdABC and flanking regions were determined. In the deduced amino acid sequence of the large subunit of the terminal oxygenase component (CbdA), a conserved motif proposed to bind the Rieske-type [2Fe-2S] cluster was identified. In the NADH:acceptor reductase component (CbdC), a putative binding site for a chloroplast-type [2Fe-2S] center and possible flavin adenine dinucleotide- and NAD-binding domains were identified. The cbdABC sequences show significant homology to benABC, which encode benzoate 1,2-dioxygenase from Acinetobacter calcoaceticus (52% identity at the deduced amino acid level), and to xylXYZ, which encode toluate 1,2-dioxygenase from Pseudomonas putida mt-2 (51% amino acid identity). Recombinant pkT231 harboring cbdABC and flanking regions complemented a plasmid-free mutant of wild-type P. cepacia 2CBS for growth on 2-chlorobenzoate, and it also allowed recombinant P. putida KT2440 to metabolize 2-chlorobenzoate. Functional NADH:acceptor reductase and oxygenase components of 2-halobenzoate 1,2-dioxygenase were enriched from recombinant Pseudomonas clones.     

Synthesis and biotransformation of 2-alkyl-4(1<Emphasis Type="Italic">H</Emphasis>)-quinolones by recombinant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2440

2-Alkyl-4(1H)-quinolones (AQs) and related derivatives, which exhibit a variety of biological properties, are secondary metabolites produced by, e.g., Pseudomonas and Burkholderia spp. Due to their main role as signaling molecules in the quorum sensing system of Pseudomonas aeruginosa, 2-heptyl-4(1H)-quinolone (HHQ) and its 3-hydroxy derivative, termed the “Pseudomonas quinolone signal” (PQS), have received considerable attention. Since chemical synthesis of different AQs is complex, we assessed the applicability of recombinant P. putida KT2440 strains for the biosynthetic production of AQs. In mineral salts medium supplemented with octanoate and anthranilate, batch cultures of P. putida KT2440 [pBBR-pqsABCD] produced about 45 μM HHQ, 30% and 70% of which were localized in the culture supernatant and methanolic cell extract, respectively. 2,4-Dihydroxyquinoline and minor amounts of C₃- to C₁₃-saturated and C_7:1 to C_13:1 monounsaturated AQs were formed as by-products. Mass spectrometry and nuclear magnetic resonance analyses spectroscopy indicated that unsaturated AQs having the same molecular mass are cis and trans isomers rather than position isomers, with the double bond located between the α and β carbon of the alkyl chain. Supplementing the cultures with hexanoate instead of octanoate shifted the AQ profile towards increased formation of C₅-AQ. Individual AQs can be prepared from concentrated methanolic extracts by preparative high-performance liquid chromatography (HPLC). Regioselective hydroxylation of HHQ to PQS can be achieved in >90% yield by biotransformation with P. putida KT2440 [pBBR-pqsH]. PQS can be isolated from methanolic cell extracts by HPLC, or be precipitated as Fe(III)-PQS complex. Preparation of a library of AQs will facilitate studies on the biological functions of these compounds.     

Purification and characterization of a fix ABCX-linked 2[4Fe-4S] ferredoxin from Azotobacter vinelandii

 Ferredoxins that contain 2[4Fe-4S]^2+/+ clusters can be divided into two classes. The "clostridial-type" ferredoxins have two CysXXCysXXCysXXXCysPro motifs. The "photosynthetic bacterial and nif-related" ferredoxins have one motif of that type and one more unusual CysXXCysX_7–9CysXXXCysPro motif. In Azotobacter vinelandii three gene sequences have been reported that contain the latter motif, but until now none of the gene products has been purified. Here we report the purification of a small anionic [Fe-S] protein with yields of ∼3 mg per 500 g cell paste. NH₂-terminal sequence analysis shows that this protein is the product of a previously sequenced A. vinelandii gene that is found upstream of fixA and is cotranscribed with fixABCX. That gene was originally named fixP, but since that gene designation is now commonly used for a very different cb-type cytochrome oxidase we have renamed the gene fixFd and its product Fix Fd. Its sequence places Fix Fd in the class of "photosynthetic bacterial and nif-related" 2[4Fe-4S]^2+/1+ ferredoxins that includes Chromatium vinosum ferredoxin. Studies of the purified protein by Fe analysis, absorption, CD and EPR spectroscopies and electrochemistry confirm this characterization; the reduction potentials of the two clusters are –440 mV vs SHE. The fact that A. vinelandii synthesizes three different proteins with the same sequence motif, each of which is likely to have a different function, shows that although sequence motifs may be used reliably to classify ferredoxins by cluster type they cannot yet be used reliably for classifying ferredoxins by function. Received: 31 January 1997 / Accepted: 9 June 1997     

Functional expression of the quinoline 2-oxidoreductase genes (qorMSL) in Pseudomonas putida KT2440 pUF1 and in P. putida 86-1 deltaqor pUF1 and analysis of the Qor proteins.

The availability of a system for the functional expression of genes coding for molybdenum hydroxylases is a prerequisite for the construction of enzyme variants by mutagenesis. For the expression cloning of quinoline 2-oxidoreductase (Qor) from Pseudomonas putida 86--that contains the molybdopterin cytosine dinucleotide molybdenum cofactor (Mo-MCD), two distinct [2Fe-2S] clusters and FAD--the qorMSL genes were inserted into the broad host range vector, pJB653, generating pUF1. P. putida KT2440 and P. putida 86-1 deltaqor were used as recipients for pUF1. Whereas Qor from the wild-type strain showed a specific activity of 19-23 U x mg(-1), the specific activity of Qor purified from P. putida KT2440 pUF1 was only 0.8-2.5 U x mg(-1), and its apparent k(cat) (quinoline) was about ninefold lower than that of wild-type Qor. The apparent Km values for quinoline were similar for both proteins. UV/visible and EPR spectroscopy indicated the presence of the full set of [2Fe-2S] clusters and FAD in Qor from P. putida KT2440 pUF1, however, the very low intensity of the Mo(V)-rapid signal, that occurs in the presence of quinoline, as well as metal analysis indicated a deficiency of the molybdenum center. In contrast, the metal content, and the spectroscopic and catalytic properties of Qor produced by P. putida 86-1 deltaqor pUF1 were essentially like those of wild-type Qor. Release of CMP upon acidic hydrolysis of the Qor proteins suggested the presence of the MCD form of the pyranopterin cofactor; the CMP contents of the three enzymes were similar.     

Evidence for the existence of [2Fe-2S] as well as [4Fe-4S] clusters among FA,FB and FX. Implications for the structure of the Photosystem I reaction center

Core extrusion of the bound iron-sulfur centers from spinach Photosystem I showed the presence of [2Fe-2S] clusters as well as [4Fe-4S] clusters among F_A, F_B and F_X. Extrusion of the iron-sulfur ensemble was not quantitative; however, the presence of [2Fe-2S] clusters correlated with higher concentration of unfolding solvent. Since F_X is highly resistant to denaturation, and since F_A and F_B are known to contain [4Fe-4S] clusters, the [2Fe-2S] clusters are assigned to F_X. The presence of [2Fe-2S] clusters in Photosystem I has significance in the structure and organization of F_X on the reaction center. Since four cysteinyl ligands are assumed to hold an iron-sulfur cluster, a Photosystem I subunit may consist of two approx. 64-kDa proteins bridged by a single [2Fe-2S] cluster. The complete reaction center would consist of two subunits positioned so that two [2Fe-2S] clusters are in magnetic interaction, thereby constituting F_X.     

Crystal structures of ferricyanide-oxidized [Fe-S] clusters in Azotobacter vinelandii ferredoxin I

Crystals of Azotobacter vinelandii ferredoxin I (FdI) have been soaked in solutions containing K₃Fe(CN)₆ in order to study the oxidation of the [3Fe-4S] and [4Fe-4S] clusters in the protein. Ferricyanide treatment results in partial loss of Fe and S from each cluster accompanied by alteration of Fe-S bonds. The effects of oxidation can be quantitated by crystallographic refinement when each [Fe-S] cluster is modeled as having a single, average structure with non-standard geometry. The oxidized clusters refined at 2.1-Å resolution display statistically significant deviations from geometric ideality. If interpreted in terms of atomic shifts these deviations indicate that each cluster first loses an inorganic S atom. In each case an Fe atom bonded to this S separates from the remaining atoms of the cluster such that the [3Fe-4S] and [4Fe-4S] clusters partially decompose into a single Fe plus 2Fe and 3Fe fragments. The extent of structural changes observed are essentially the same in crystals soaked at 3?:?1, 9?:?1 and 30?:?1 mole ratio of K₃ Fe(CN)₆?:?FdI, suggesting that the crystal lattice permits limited oxidation reactions to occur at a low mole ratio but restricts conformational changes from occurring that may be required for more extensive oxidative reactions at higher mole ratio. The results are relevant to understanding the transformations which may take place when [Fe-S] proteins are deliberately oxidized with ferricyanide.     

Glutathione-complexed [2Fe-2S] clusters function in Fe–S cluster storage and trafficking

Glutathione-coordinated [2Fe-2S] complex is a non-protein-bound [2Fe-2S] cluster that is capable of reconstituting the human iron-sulfur cluster scaffold protein IscU. This complex demonstrates physiologically relevant solution chemistry and is a viable substrate for iron-sulfur cluster transport by Atm1p exporter protein. Herein, we report on some of the possible functional and physiological roles for this novel [2Fe-2S](GS₄) complex in iron-sulfur cluster biosynthesis and quantitatively characterize its role in the broader network of Fe–S cluster transfer reactions. UV–vis and circular dichroism spectroscopy have been used in kinetic studies to determine second-order rate constants for [2Fe-2S] cluster transfer from [2Fe-2S](GS₄) complex to acceptor proteins, such as human IscU, Schizosaccharomyces pombe Isa1, human and yeast glutaredoxins (human Grx2 and Saccharomyces cerevisiae Grx3), and human ferredoxins. Second-order rate constants for cluster extraction from these holo proteins were also determined by varying the concentration of glutathione, and a likely common mechanism for cluster uptake was determined by kinetic analysis. The results indicate that the [2Fe-2S](GS₄) complex is stable under physiological conditions, and demonstrates reversible cluster exchange with a wide range of Fe–S cluster proteins, thereby supporting a possible physiological role for such centers.     

Redox centers of 4-hydroxybenzoyl-CoA reductase, a member of the xanthine oxidase family of molybdenum-containing enzymes

4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a key enzyme in the anaerobic metabolism of phenolic compounds. It catalyzes the reductive removal of the hydroxyl group from the aromatic ring yielding benzoyl-CoA and water. The subunit architecture, amino acid sequence, and the cofactor/metal content indicate that it belongs to the xanthine oxidase (XO) family of molybdenum cofactor-containing enzymes. 4-HBCR is an unusual XO family member as it catalyzes the irreversible reduction of a CoA-thioester substrate. A radical mechanism has been proposed for the enzymatic removal of phenolic hydroxyl groups. In this work we studied the spectroscopic and electrochemical properties of 4-HBCR by EPR and M?ssbauer spectroscopy and identified the pterin cofactor as molybdopterin mononucleotide. In addition to two different [2Fe-2S] clusters, one FAD and one molybdenum species per monomer, we also identified a [4Fe-4S] cluster/monomer, which is unique among members of the XO family. The reduced [4Fe-4S] cluster interacted magnetically with the Mo(V) species, suggesting that the centers are in close proximity, (<15 A apart). Additionally, reduction of the [4Fe-4S] cluster resulted in a loss of the EPR signals of the [2Fe-2S] clusters probably because of magnetic interactions between the Fe-S clusters as evidenced in power saturation studies. The Mo(V) EPR signals of 4-HBCR were typical for XO family members. Under steady-state conditions of substrate reduction, in the presence of excess dithionite, the [4Fe-4S] clusters were in the fully oxidized state while the [2Fe-2S] clusters remained reduced. The redox potentials of the redox cofactors were determined to be: [2Fe-2S](+1/+2) I, -205 mV; [2Fe-2S] (+1/+2) II, -255 mV; FAD/FADH( small middle dot)/FADH, -250 mV/-470 mV; [4Fe-4S](+1/+2), -465 mV and Mo(VI)/(V)/(VI), -380 mV/-500 mV. A catalytic cycle is proposed that takes into account the common properties of molybdenum cofactor enzymes and the special one-electron chemistry of dehydroxylation of phenolic compounds.     

The F420-Reducing [NiFe]-Hydrogenase Complex from Methanothermobacter marburgensis, the First X-ray Structure of a Group 3 Family Member

The reversible redox reaction between coenzyme F₄₂₀ and H₂ to F₄₂₀H₂ is catalyzed by an F₄₂₀-reducing [NiFe]-hydrogenase (FrhABG), which is an enzyme of the energy metabolism of methanogenic archaea. FrhABG is a group 3 [NiFe]-hydrogenase with a dodecameric quaternary structure of 1.25 MDa as recently revealed by high-resolution cryo-electron microscopy. We report on the crystal structure of FrhABG from Methanothermobacter marburgensis at 1.7 Å resolution and compare it with the structures of group 1 [NiFe]-hydrogenases, the only group structurally characterized yet. FrhA is similar to the large subunit of group 1 [NiFe]-hydrogenases regarding its core structure and the embedded [NiFe]-center but is different because of the truncation of ca 160 residues that results in similar but modified H₂ and proton transport pathways and in suitable interfaces for oligomerization. The small subunit FrhG is composed of an N-terminal domain related to group 1 enzymes and a new C-terminal ferredoxin-like domain carrying the distal and medial [4Fe-4S] clusters. FrhB adopts a novel fold, binds one [4Fe-4S] cluster as well as one FAD in a U-shaped conformation and provides the F₄₂₀-binding site at the Si-face of the isoalloxazine ring. Similar electrochemical potentials of both catalytic reactions and the electron-transferring [4Fe-4S] clusters, determined to be E°′ ≈ − 400 mV, are in full agreement with the equalized forward and backward rates of the FrhABG reaction. The protein might contribute to balanced redox potentials by the aspartate coordination of the proximal [4Fe-4S] cluster, the new ferredoxin module and a rather negatively charged FAD surrounding.     

Crystal structure of the 100 kDa arsenite oxidase from Alcaligenes faecalis in two crystal forms at 1.64 A and 2.03 A

BACKGROUND: Arsenite oxidase from Alcaligenes faecalis NCIB 8687 is a molybdenum/iron protein involved in the detoxification of arsenic. It is induced by the presence of AsO(2-) (arsenite) and functions to oxidize As(III)O(2-), which binds to essential sulfhydryl groups of proteins and dithiols, to the relatively less toxic As(V)O(4)(3-) (arsenate) prior to methylation. RESULTS: Using a combination of multiple isomorphous replacement with anomalous scattering (MIRAS) and multiple-wavelength anomalous dispersion (MAD) methods, the crystal structure of arsenite oxidase was determined to 2.03 A in a P2(1) crystal form with two molecules in the asymmetric unit and to 1.64 A in a P1 crystal form with four molecules in the asymmetric unit. Arsenite oxidase consists of a large subunit of 825 residues and a small subunit of approximately 134 residues. The large subunit contains a Mo site, consisting of a Mo atom bound to two pterin cofactors, and a [3Fe-4S] cluster. The small subunit contains a Rieske-type [2Fe-2S] site. CONCLUSIONS: The large subunit of arsenite oxidase is similar to other members of the dimethylsulfoxide (DMSO) reductase family of molybdenum enzymes, particularly the dissimilatory periplasmic nitrate reductase from Desulfovibrio desulfuricans, but is unique in having no covalent bond between the polypeptide and the Mo atom. The small subunit has no counterpart among known Mo protein structures but is homologous to the Rieske [2Fe-2S] protein domain of the cytochrome bc(1) and cytochrome b(6)f complexes and to the Rieske domain of naphthalene 1,2-dioxygenase.     

AI-2通过甲基化趋化受体McpU调控恶臭假单胞菌KT2440的趋化运动及生物膜形成

[背景]广泛存在于革兰氏阴性菌和革兰氏阳性菌中的自诱导物autoinducer-2 (AI-2)能够介导细菌种内和种间通讯,并调节细菌的多种生理过程.然而恶臭假单胞菌KT2440能否感知AI-2信号还未见报道.[目的]挖掘介导恶臭假单胞菌KT2440对AI-2趋化反应的趋化受体,检测AI-2信号通过趋化受体对恶臭假单胞...     

Iron-sulfur interconversions in the anaerobic ribonucleotide reductase from Escherichia coli

The anaerobic ribonucleotide reductase from Escherichia coli contains an iron-sulfur cluster which, in the reduced [4Fe-4S]⁺ form, serves to reduce S-adenosylmethionine and to generate a catalytically essential glycyl radical. The reaction of the reduced cluster with oxygen was studied by UV-visible, EPR, NMR, and Mössbauer spectroscopies. The [4Fe-4S]⁺ form is shown to be extremely sensitive to oxygen and converted to [4Fe-4S]²⁺, [3Fe-4S]^+/0, and to the stable [2Fe-2S]²⁺ form. It is remarkable that the oxidized protein retains full activity. This is probably due to the fact that during reduction, required for activity, the iron atoms, from 2Fe and 3Fe clusters, readily reassemble to generate an active [4Fe-4S] center. This property is discussed as a possible protective mechanism of the enzyme during transient exposure to air. Futhermore, the [2Fe-2S] form of the protein can be converted into a [3Fe-4S] form during chromatography on dATP-Sepharose, explaining why previous preparations of the enzyme were shown to contain large amounts of such a 3Fe cluster. This is the first report of a 2Fe to 3Fe cluster conversion.     

Structure and electrochemistry of proteins harboring iron-sulfur clusters of different nuclearities. Part IV. Canonical,non-canonical and hybrid iron-sulfur proteins

A plethora of proteins are able to express iron-sulfur clusters, but have a clear picture of the different types of proteins and the different iron-sulfur clusters they harbor it is not easy.In the last five years we have reviewed structure/electrochemistry of metalloproteins expressing: (i) single types of iron-sulfur clusters (namely: {Fe(Cys)₄}, {[Fe₂S₂](Cys)₄}, {[Fe₂S₂](Cys)₃(X)} (X?=?Asp, Arg, His), {[Fe₂S₂](Cys)₂(His)₂}, {[Fe₃S₄](Cys)₃}, {[Fe₄S₄](Cys)₄} and {[Fe₄S₄](Cys)₃(nonthiolate ligand)} cores); (ii) metalloproteins harboring iron-sulfur centres of different nuclearities (namely: [4Fe-4S] and [2Fe-2S], [4Fe-4S] and [3Fe-4S], and [4Fe-4S], [3Fe-4S] and [2Fe-2S] clusters. Our target is now to review structure and electrochemistry of proteins harboring canonical, non-canonical and hybrid iron-sulfur proteins.     

Novel structure and redox chemistry of the prosthetic groups of the iron-sulfur flavoprotein sulfide dehydrogenase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis>; evidence for a [2Fe-2S] cluster with Asp(Cys)<Subscript>3</Subscript> ligands

The consecutive structural genes for the iron-sulfur flavoenzyme sulfide dehydrogenase, sudB and sudA, have been identified in the genome of Pyrococcus furiosus. The translated sequences encode a heterodimeric protein with an alpha-subunit, SudA, of 52598 Da and a beta-subunit, SudB, of 30686 Da. The alpha-subunit carries a FAD, a putative nucleotide binding site for NADPH, and a [2Fe-2S]2+,+ prosthetic group. The latter exhibit EPR g-values, 2.035, 1.908, 1.786, and reduction potential, Em,8 = +80 mV, reminiscent of Rieske-type clusters; however, comparative sequence analysis indicates that this cluster is coordinated by a novel motif of one Asp and three Cys ligands. The motif is not only found in the genome of hyperthermophilic archaea and hyperthermophilic bacteria, but also in that of mesophilic Treponema pallidum. The beta-subunit of sulfide dehydrogenase contains another FAD, another putative binding site for NADPH, a [3Fe-4S]+,0 cluster, and a [4Fe-4S]2+,+ cluster. The 3Fe cluster has an unusually high reduction potential, Em,8 = +230 mV. The reduced 4Fe cluster exhibits a complex EPR signal, presumably resulting from magnetic interaction of its S = 1/2 spin with the S=2 spin of the reduced 3Fe cluster. The 4Fe cluster can be reduced with deazaflavin/EDTA/light but not with sodium dithionite; however, it is readily reduced with NADPH. SudA is highly homologous to KOD1-GO-GAT (or KOD1-GltA), a single-gene encoded protein in Pyrococcus kodakaraensis, which has been putatively identified as hyperthermophilic glutamate synthase. However, P. furiosus sulfide dehydrogenase does not have glutamate synthase activity. SudB is highly homologous to HydG, the gamma-subunit of P. furiosus NiFe hydrogenase. The latter enzyme also has sulfide dehydrogenase activity. The P. furiosus genome contains a second set of consecutive genes, sudY and sudX, with very high homology to the sudB and sudA genes, and possibly encoding a sulfide dehydrogenase isoenzyme. Each subunit of sulfide dehydrogenase is a primary structural paradigm for a different class of iron-sulfur flavoproteins.     

Purification and characterization of salicylate 5-hydroxylase, a three-component monooxygenase from Ralstonia sp. strain U2

Salicylate is an important intermediate in the bacterial degradation of polycyclic aromatic hydrocarbons and salicylate hydroxylases play essential roles in linking the peripheral and ring-cleavage catabolic pathways. Unlike the well-characterized salicylate 1-hydroxylases, the rarely occurred salicylate 5-hydroxylase (S5H) has not been characterized in detail. In this study, the three-component Fe-S protein complex (NagAaGHAb) of S5H from Ralstonia sp. strain U2 was purified, and its biochemical and catalytic properties were characterized. The oxygenase component NagGH exhibited an α₃β₃ heterohexameric structure and contained one Rieske-type [2Fe-2S] cluster and one mononuclear iron per α subunit. NagAa is the ferredoxin-NADP⁺ reductase component containing flavin and plant type [2Fe–2S] cluster. The ferredoxin component NagAb was characterized as a [2Fe-2S] dimer which remains remarkably stable in denaturing gel electrophoresis after being heated at 100 °C for 1 h. Purified NagAa and NagAb, NagGH catalyzed the hydroxylation of salicylate to gentisate with a specific activity of 107.12?±?14.38 U/g and showed an apparent K _m for salicylate of 102.79?±?27.20 μM and a similar K _m value for both NADH and NADPH (59.76?±?7.81 μM versus 56.41?±?12.76 μM). The hydroxylase exhibited different affinities for two hydroxysalicylates (2,4-dihydroxybenzoate K _m of 93.54?±?18.50 μM versus 2,6-dihydroxybenzoate K _m of 939.80?±?199.46 μM). Interestingly, this S5H also showed catalytic activity to the pollutant 2-nitrophenol and exhibited steady-state kinetic data of the same order of magnitude as those for salicylate. This study will allow further comparative studies of structure–function relationships of the ring hydroxylating mono- and di-oxygenase systems.     

Efficient hydroxylation of 1,8-cineole with monoterpenoid-resistant recombinant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> GS1

In this work, monoterpenoid hydroxylation with Pseudomonas putida GS1 and KT2440 were investigated as host strains, and the cytochrome P450 monooxygenase CYP176A1 (P450_cin) and its native redox partner cindoxin (CinC) from Citrobacter braakii were introduced in P. putida to catalyze the stereoselective hydroxylation of 1,8-cineole to (1R)-6β-hydroxy-1,8-cineole. Growth experiments in the presence of 1,8-cineole confirmed pseudomonads’ superior resilience compared to E. coli. Whole-cell P. putida harboring P450_cin with and without CinC were capable of hydroxylating 1,8-cineole, whereas coexpression of CinC has been shown to accelerate this bioconversion. Under the same conditions, P. putida GS1 produced more than twice the amount of heterologous P450_cin and bioconversion product than P. putida KT2440. A concentration of 1.1 ± 0.1 g/L (1R)-6β-hydroxy-1,8-cineole was obtained within 55 h in shake flasks and 13.3 ± 1.9 g/L in 89 h in a bioreactor, the latter of which corresponds to a yield Y_P/S of 79 %. To the authors’ knowledge, this is the highest product titer for a P450 based whole-cell monoterpene oxyfunctionalization reported so far. These results show that solvent-tolerant P. putida GS1 can be used as a highly efficient recombinant whole-cell biocatalyst for a P450 monooxygenase-based valorization of monoterpenoids.     

Modular organization and identification of a mononuclear iron-binding site within the NifU protein

The NifS and NifU nitrogen fixation-specific gene products are required for the full activation of both the Fe-protein and MoFe-protein of nitrogenase from Azotobacter vinelandii. Because the two nitrogenase component proteins both require the assembly of [Fe-S]-containing clusters for their activation, it has been suggested that NifS and NifU could have complementary functions in the mobilization of sulfur and iron necessary for nitrogenase-specific [Fe-S] cluster assembly. The NifS protein has been shown to have cysteine desulfurase activity and can be used to supply sulfide for the in vitro catalytic formation of [Fe-S] clusters. The NifU protein was previously purified and shown to be a homodimer with a [2Fe-2S] cluster in each subunit. In the present work, primary sequence comparisons, amino acid substitution experiments, and optical and resonance Raman spectroscopic characterization of recombinantly produced NifU and NifU fragments are used to show that NifU has a modular structure. One module is contained in approximately the N-terminal third of NifU and is shown to provide a labile rubredoxin-like ferric-binding site. Cysteine residues Cys³⁵, Cys⁶², and Cys¹⁰⁶ are necessary for binding iron in the rubredoxin-like mode and visible extinction coefficients indicate that up to one ferric ion can be bound per NifU monomer. The second module is contained in approximately the C-terminal half of NifU and provides the [2Fe-2S] cluster-binding site. Cysteine residues Cys¹³⁷, Cys¹³⁹, Cys¹⁷², and Cys¹⁷⁵ provide ligands to the [2Fe-2S] cluster. The cysteines involved in ligating the mononuclear Fe in the rubredoxin-like site and those that provide the [2Fe-2S] cluster ligands are all required for the full physiological function of NifU. The only two other cysteines contained within NifU, Cys²⁷² and Cys²⁷⁵, are not necessary for iron binding at either site, nor are they required for the full physiological function of NifU. The results provide the basis for a model where iron bound in labile rubredoxin-like sites within NifU is used for [Fe-S] cluster formation. The [2Fe-2S] clusters contained within NifU are proposed to have a redox function involving the release of Fe from bacterioferritin and/or the release of Fe or an [Fe-S] cluster precursor from the rubredoxin-like binding site. Received: 27 October 1999 / Accepted: 30 November 1999