Earticle

CYP105D7, cytochrome p450 from Streptomyces avermitilis was cloned using T7 promoter of the bacterial expression vector pET24ma. Expressed CYP105D7 in E.coli BL21, convert daidzein to 7,3’4’-trihydroxyisoflavone in-vivo and in-vitro system using NADH-dependant reducing equivalent ferredoxin reductase (PDR) and ferredoxin (PDX) from pseudomonas putida.The hydroxylated position was confirmed by GC-MS analysis. The total turnover number of the enzyme was 0.69μmol 7,3’4’-trihydroxyisoflavone produced per μmol p450 per min.To overproduce the 3’ODI using this novel enzyme ,self host Streptomyces avermitilis were used. Recombinant Streptomyces avermitilis strain was found to have 3.6 times higher activity than the wild type to produce 7,3’4’-trihydroxyisoflavone. The cytochrome p450 CYP105D7 of Streptomyces avermitilis was detected by CO-difference spectra in whole cell suspension. Large scale recombinant Streptomyces avermitilis shows conversion of 381 mg dadizein using 7 –l-jar fermentor yielded 112.5 mg of 7,3’4’-trihydroxyisoflavone.

Ferredoxin-NADP+ reductase (Fpr) is known to control NADP+/NADPH pool in bacteria. Interestingly, P. putida have two Fpr (FprA, FprB) and its two cognate [2Fe-2S] Ferredoxins (FdA, FdB). Kinetic study of each Fpr was performed using a variety of assays [diaphorase, cytochromeC and ferric reductase assay]. Yeast two-hybrid was used to determine in vivo interaction between Fpr and Fd. Likewise Fpr of other bacteria, both FprA and FprB can use DPIP and ferricyanide as the terminal electron acceptor, which indicated that both Fprs have diaphorase activity. We confirmed that both Fpr products prefer NADPH to NADH at the redox complex formation. In the cytochromeC and ferric reductase assay, complex between FprB and FdA has high catalytic activity (kcat/Km) which is 5-fold higher than that of other redox couples. Similarly, In vivo interaction study using yeast two hybrid system confirms that FprB has more specific binding activity with FdA than with FdB. The data shows that specific interaction between Fpr and its cognate partner is required for efficient catalytic function. This work was supported by a NCRC grant (R15-2003-012-02002-0) and a grant (R0503443) from the BioGreen21 program

Proliferating cell nuclear antigen (PCNA) is a trimeric ring‐shaped protein that binds to dsDNA and acts as a scaffold for DNA‐related enzymes, such as DNA polymerase and helicase. Although most of PCNAs form homotrimers, three PCNAs found in Sulfolobus solfataricus form a heterotrimer. Fusion proteins between these PCNAs and functional proteins can act as nano‐scale parts, which can self‐assemble to form functional nano‐hybrid complex. Bacterial cytochrome P450 (P450), which is a promising biocatalyst, generally needs to accept electrons from NAD(P)H for its monooxygenase activity, interacting with a ferredoxin that is reduced by a specific ferredoxin reductase with NAD(P)H. Fusing these proteins with PCNAs can generate functional stand‐alone complexes of P450 and electron transfer related proteins. Here we show that three PCNA proteins, fused with either P450 from Pseudomonas putida (P450cam) or one of two electron transfer related proteins (Pdx, PdR), formed a stable heterotrimeric complex (PCNA‐Utilized Protein Complex of P450 and Its Two Electron Transfer Related Proteins, PUPPET). In PUPPET, P450cam, PdX and PdR were in extremely close proximity to each other, enabling efficient electron transfer within the complex (Fig. 1). As a result, PUPPET showed about 60‐fold higher catalytic activities compared with an equimolar mixture of the constituent components. The formation of complexes utilizing PCNA could be a valuable strategy for the construction of complicated multi‐enzymatic reactions, as well as for applications using electron transfer related proteins.

The filed of molecular electronics has seen an outburst of interest particularly motivated by the possibility of developing and characterizing the individual molecules to perform functions in electronic circuitry as performed by semiconductor devices[1-3]. Towards this goal, we have developed a molecular memory device that includes novel immobilization method for ferredoxin on gold surface. A recombinant protein with cysteine residue by using Polymerase Chain Reaction (PCR) was designed and then directly immobilized on Au surface without any chemical linker. The immobilization of the functionalized protein is confirmed by Surface Plasmon Resonance (SPR) and its surface morphology is analyzed by Scanning Tunneling Microscopy (STM). The memory function of self assembled ferredoxin was investigated by its redox properties of metalloprotein using Cyclic voltammetry, Chronoamperometry and Open Circuit Potential Amperometry (OCPA).The approaches outlined here can be applied for the development of the molecular memory device

The coenzyme A-acylating 2-oxoacid:ferredoxin oxidoreductase and ferredoxin (an effective electron acceptor) were purified from the hyperthermophilic archaeon, Sulfolobus solfataricus P1 (DSM1616). The purified ferredoxin is a monomeric protein with an apparent molecular mass of approximately 11 kDa by SDS-PAGE and of $11,180{\pm}50$ Da by MALDI-TOF mass spectrometry. Ferredoxin was identified to be a dicluster, [3Fe-4S][4Fe-4S], type ferredoxin by spectrophotometric and EPR studies, and appeared to be zinc-containing based on the shared homology of its N-terminal sequence with those of known zinc-containing ferredoxins. On the other hand, the purified 2-oxoacid: ferredoxin oxidoreductase was found to be a heterodimeric enzyme consisting of 69 kDa $\alpha$ and 34 kDa $\beta$ subunits by SDS-PAGE and MALDI-TOF mass spectrometry. The purified enzyme showed a specific activity of 52.6 units/mg for the reduction of cytochrome c with 2-oxoglutarate as substrate at $55^{\circ}C$, pH 7.0. Maximum activity was observed at $70^{\circ}C$ and the optimum pH for enzymatic activity was 7.0 -8.0. The enzyme displays broad substrate specificity toward 2-oxoacids, such as pyruvate, 2-oxobutyrate, and 2-oxoglutarate. Among the 2-oxoacids tested (pyruvate, 2-oxobutyrate, and 2-oxoglutarate), 2-oxoglutarate was found to be the best substrate with $K_m$ and $k_{cat}$ values of $163\;{\mu}M$ and $452\;min^{-1}$, respectively. These results provide useful information for structural studies on these two proteins and for studies on the mechanism of electron transfer between the two.

Pseudomonas sp. S-47 is capable of degrading catechol and 4-chlorocatechol via the meta-cleavage pathway. XyITE products catalyze the dioxygenation of the aromatics. The sylT of the strain S-47 is located just upstream of the xylE gene. XylT of the strain S-47 is located just upstream of the xylE gene. XyIT is typical chloroplast-type ferredoxin, which is characterized by 4 cystein residues that are located at positions 41, 46, 49, and 81. The chloroplast-type ferredoxin of Pseudomonas sp. S-47 exhibited a 98% identity with that of P. putida mt-2(TOL plasmid) in the amino acid sequence, but only about a 40 to 60% identity with the corresponding enzymes from other organisms. We constructed two recombinant plasmids (pRES1 containing xylTE and pRES101 containing xylE without xylT) in order to examine the function of XyIT for the reactivation of the catechol 2,3-dioxygenase (XyIE) that is oxidized with hydrogen peroxide was recovered in the catechol 2,3-dioxygenase (C23O) activity about 4 mimutes after incubation, but the pRES101 showed no recovery. That means that the typical chloroplast-type ferredoxin (XyIT) of Pseudomonas sp. S-47 is involved in the reactivation of the oxidized C23O in the dioxygenolytic cleavage of aromatic compounds.

Flavodoxin (Fld) has been demonstrated to bind to ferredoxin-NADP$^+$ reductase A (FprA) in Pseudomonas putida. Two residues ($Phe^{256}$, $Lys^{259}$) of FprA are likely to be important for interacting with Fld based on homology modeling. Site-directed mutagenesis and pH-dependent enzyme kinetics were performed to further examine the role of these residues. The catalytic efficiencies of FprA-$Ala^{259}$ and FprA-$Asp^{259}$ proteins were two-fold lower than those of the wild-type FprA. Homology modeling also strongly suggested that these two residues are important for electron transfer. Thermodynamic properties such as entropy, enthalpy, and heat capacity changes of FprA-$Ala^{259}$ and FprA-$Asp^{259}$ were examined by isothermal titration calorimetry. We demonstrated, for the first time, that $Phe^{256}$ and $Lys^{259}$ are critical residues for the interaction between FprA and Fld. Van der Waals interactions and hydrogen bonding were also more important than ionic interactions for forming the FprA-Fld complex.

The fpr gene, which encodes a ferredoxin-$NADP^+$ reductase, is known to participate in the reversible redox reactions between $NADP^+$/NADPH and electron carriers, such as ferredoxin or flavodoxin. The role of Fpr and its regulatory protein, FinR, in Pseudomonas putida KT2440 on the oxidative and osmotic stress responses has already been characterized [Lee at al. (2006). Biochem. Biophys. Res. Commun. 339, 1246-1254]. In the genome of P. putida KT2440, another Fpr homolog (FprB) has a 35.3% amino acid identity with Fpr. The fprB gene was cloned and expressed in Escherichia coli. The diaphorase activity assay was conducted using purified FprB to identify the function of FprB. In contrast to the fpr gene, the induction of fprB was not affected by oxidative stress agents, such as paraquat, menadione, $H_2O_2$, and t-butyl hydroperoxide. However, a higher level of fprB induction was observed under osmotic stress. Targeted disruption of fprB by homologous recombination resulted in a growth defect under high osmotic conditions. Recovery of oxidatively damaged aconitase activity was faster for the fprB mutant than for the fpr mutant, yet still slower than that for the wild type. Therefore, these data suggest that the catalytic function of FprB may have evolved to augment the function of Fpr in P. putida KT2440.

Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.

A ferredoxin adsorbed hetero self-assembled layer was fabricated on chemically modified Au substrate, 4-Aminothiophenol (4-ATP) was deposited onto Au substrate and then N-succinimidyl-3-[2-pyridyldithio] propionate (SPDP) was adsorbed on the 4-ATP layer, since SPDP was used as a bridging molecule for ferredoxin adsorption, Ferredoxin/SPDP/4-ATP structured hetero layer was constructed because of strong chemical binding of ferredoxin, SPDP, and 4-ATP, The surface of the ferredoxin-adsorbed SPDP/4-ATP layer was observed by scanning tunneling microscopy, The hetero film formation was verified by surface plasmon resonance measurement. The current flow and rectifying property based on the scanning tunneling spectroscopy I-V characteristics was achieved in the proposed hetero layer. Thus, the hetero layer structure of ferredoxin functioned as a molecular diode with rectifying property, The proposed molecular diode can be usefully applied for the development of molecular scale electronic devices.

The stability and electrochemical properties of a self-assembled layer of spinach ferredoxin on a quartz substrate and on a highly oriented pyrolytic graphite electrode were investigated. To fabricate the ferredoxin self-assembly layer, dimyristoylphosphatidylcholine was first deposited onto a substrate for ferredoxin immobilization. Surface analysis of the ferredoxin layer was carried out by atomic force microscopy to verify the ferredoxin immobilization. To verify ferredoxin immobilization on the lipid layer and to confirm the maintenance of redox activity, absorption spectrum measurement was carried out. Finally, cyclic-voltammetry measurements were performed on the ferredoxin layers and the redox potentials were obtained. The redox potential of immobilized ferredoxin had a formal potential value of -540 mV. It is suggested that the redox-potential measurement of self-assembled ferredoxin molecules could be used to construct a biosensor and biodevice.

Ferredoxin is a typical iron-sulfur protein that is ubiquitous in biological redox systems. This study investigates the in vitro assembly of a [$Fe_2S_2$] cluster in the ferredoxin from Acidithiobacillus ferrooxidans in the presence of three scaffold proteins: IscA, IscS, and IscU. The spectra and MALDI-TOF MS results for the reconstituted ferredoxin confirm that the iron-sulfur cluster was correctly assembled in the protein. The inactivation of cysteine desulfurase by L-allylglycine completely blocked any [$Fe_2S_2$] cluster assembly in the ferredoxin in E. coli, confirming that cysteine desulfurase is an essential component for iron-sulfur cluster assembly. The present results also provide strong evidence that [$Fe_2S_2$] cluster assembly in ferredoxin follows the AUS pathway.

Pseudomonas sp. DJ77로부터 전보에서 클로닝한 pHENX7의 하류방향으로 약 3kb 정도를 포함하는 pYCS500을 클로닝하였다. PYCS500의 제한효소지도를 작성하고 부분적으로 염기서열을 분석한 결과 465 bp의 HindIII-ClaI절편에서 282 bp로 이루어진 하나의 open reading frame(ORF)을 발견하였다. phnM으로 명명된 이 ORF는 93개의 아미노산으로 구성된 polypeptide를 암호화하고 있었으며 계산된 분자량은 10,008 Da이었다. PhnM은 NahT, XylT, DmpQ, AtdS, PhlG, PhhQ, TbuW 등 plant-type ferredoxin 형태의 단백질과 37.7%-53.9%의 상동성을 나타내었으며 이들이 공통적으로 가지고 있는 motif가 일치하였다.

We cloned the 4.8 kb BglII fragment containing genes downstream pHENX7 from Pseudomonas sp. strain DJ77. The restriction map of the resultant clone, recombinant plasmid pYCS500, was determined. Sequencing analysis of the 465 bp HindIII-ClaI fragment revealed an open reading frame of 282 bp that was then designated phnM. The deduced polypeptide is 93 amino acid residues long with a $M_r$ of 10,008. The PhnM has 37.3-53.9% identity with plant-type ferredoxin proteins such as NahT, XylT, DmpQ, AtdS, PhlG, PhhQ and TbuW and contains the motif similar to well-conserved functional domains of those proteins.

One of the three components of the phenanthrene dioxygenase which is required for conversion of phenanthrene to cis-phenanthrene dihydrodiol, Rieske-type ferredoxin encoded by phnR has been cloned and sequenced from Pseudomonas sp. strain DJ77. The gene phnR is positioned at the downstream of phnQ encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase. The PhnR ferredoxin contains 108 amino acids with a Mr of 11,355. The deduced amino acid sequence of the PhnR ferredoxin is 35-79% identical to those of homologous ferredoxins encoded by various genes.

We isolated a promoter inducible by paraquat, a superoxide-generating agent, from Escherichia coli using a promoter-probing plasmid pRS415. From sequence analysis we found out the promoter is for fpr ENCODING nadph : ferredoxin oxidoreductase. We constructed on operon fusion of lacZ gene with fpr promoter to monitor the expression of the gene in the single-copy state. LacZ expression generators, menadione and plumbagin, also induced the expression of .betha.-galactosidase in the fusion strain. On the other hand, no significant induction was observed by treatment with hydrogen peroxide, ethanol, and heat shock. Induction of .betha.-galactosidase was significantly reduced by introducing a .DELTA. sox 8 :: cat of soxS3 :: Tn10 mutation into the fusion strain, indicating that fpr gene is a member of the soxRS regulon. The transcriptional start site was determined by primer extension analysis. Possible roles of fpr induction in superoxide stress were discussed.

The 1H NMR signals of the heme methyl, propionate and related chemical groups of cytochrome C3 from Desulfovibrio vulgaris Miyazaki F (D.v. MF) were assigned by means of 1D NOE, 2D DQFCOSY and 2D TOCSY spectra. They were consistent with the assignments of the hemes with the highest and second-lowest redox potentials reported by Gayda et al. [Reference: 15]. The heme assignments were also supported by NOE between the methyl groups of these hemes and the side chain of Val-18, All the results contradicted the heme assignments for D.v. MF cytochrome C3 made on the basis of NMR [Reference: 11]. Based on these assignments, the interaction of cytochrome C3 with ferredoxin I was investigated by NMR. The major interaction site of cytochrome C3 was identified as the heme with the highest redox potential, which is surrounded by the highest density of positive charges. The stoichiometry and association constant were two cytochrome C3 molecules per monomer of ferredoxin I and 108 M-2 (at 53 mM ionic strength and $25^{\circ}C$), respectively.