Heterologous expression, purification, and characterization of cytochrome P450sca-2 and mutants with improved solubility in Escherichia coli

Pravastatin, an important cholesterol lowering drug, is currently produced by hydroxylation of mevastatin (ML-236B) with Streptomyces carbophilus, in which the enzyme P450sca-2 plays a key role. Little information on the recombinant expression of this enzyme is available. As it is of industrial interest to develop an alternative simplified enzymatic process for pravastatin, as a first step, further study on the heterologous expression of this enzyme is warranted. We report here, for the first time, the purification, and characterization of P450sca-2 expressed in Escherichia coli. A synthetic gene encoding P450sca-2 was designed to suit the standard codon usage of E. coli. Expression of P450sca-2 in E. coli under optimized conditions yielded about 100 nmol purified active P450sca-2 per liter. Directed evolution was further carried out to improve the soluble expression level. In the absence of a facile and sensitive assay, green fluorescent protein (GFP) was used as a reporter to enable high-throughput screening. After three rounds of evolution by error-prone PCR and DNA shuffling, six almost totally soluble mutants were obtained, with the soluble expression levels dramatically improved by about 30-fold. For six most frequently occurring mutations, the corresponding single mutants were created to dissect the effects of these mutations. A single mutation, P159A, was found to be responsible for most of the enhanced solubility observed in the six mutants, and the corresponding single mutant also retained the hydroxylation activity. Our study provides a foundation for future work on improving functional expression of P450sca-2 in E. coli.     

Isolation and identification of a novel Rhodococcus sp. ML-0004 producing epoxide hydrolase and optimization of enzyme production

Rhodococcus sp. ML-0004, a novel strain for producing epoxide hydrolase, was isolated from soil in this study. The epoxide hydrolase can catalyze the stereo-specific hydrolysis of cis-epoxysuccinic acid to generate l(+)-tartaric acid. By examining physiological, biochemical characteristics and comparing its 16S rDNA gene sequence, it was identified as Rhodococcus opacus, and named R. opacus ML-0004. The optimal conditions for epoxide hydrolase production from R. opacus ML-0004 were also investigated. Propanediol and (NH₄)₂SO₄ were selected as carbon source and nitrogen source, respectively, for the production of R. opacus ML-0004 epoxide hydrolase. The optimal conditions for epoxide hydrolase production were fermentation temperature = 28 °C, pH 7.0, and cultivation time = 26 h. Under these conditions, the maximum epoxide hydrolase activity reached 10.5 U mL⁻¹.     

Genomic organization and promoter activity of the maize starch branching enzyme I gene

Starch branching enzymes (SBE) which catalyse the formation of α-1,6-glucan linkages are of crucial importance for the quantity and quality of starch synthesized in plants. In maize (Zea mays L.), three SBE isoforms (SBEI, IIa and IIb) have been identified and shown to exhibit differential expression patterns. As a first step toward understanding the regulatory mechanisms controlling their expression, we isolated and sequenced a maize genomic DNA (−2190 to +5929) which contains the entire coding region of SBEI (Sbe1) as well as 5′-and 3′-flanking sequences. Using this clone, we established a complete genomic organization of the maize Sbe1 gene. The transcribed region consists of 14 exons and 13 introns, distributed over 5.7 kb. A consensus TATA-box and a G-box containing a perfect palindromic sequence, CCACGTGG, were found in the 5′-flanking region. Genomic Southern blot analysis indicated that two Sbe1 genes with divergent 5′-flanking sequences exist in the maize genome, suggesting the possibility that they are differentially regulated. A chimeric construct containing the 5′-flanking region of Sbe1 (−2190 to +27) fused to the β-glucuronidase gene (pKG101) showed promoter activity after it was introduced into maize endosperm suspension cells by particle bombardment.     

Cytochrome-c-assisted escape of cardiolipin from a model mitochondrial membrane

Binding of cytochrome c (Cytc) to cardiolipin (CL) in the inner mitochondrial membrane is involved with the onset of apoptosis. In this study, we used CL-containing phospholipid monolayers to mimic the inner mitochondrial membrane. Constant pressure insertion assay was employed to monitor the Cytc-induced expansion of membrane area. Simultaneous epifluorescence microscopy imaging afforded the in-situ visualization of phospholipid demixing and sorting in the membrane. The formation of a CL-rich L_d phase has been observed to prelude the insertion of Cytc. Based on the relative expansion of membrane area, a cluster of a few amino acid residues of Cytc with an area of 117 ± 7 Å² has been found to insert into the membrane. The insertion of Cytc disrupted the membrane in a way facilitating the escape of CL. When the exclusion of Cytc was induced by compression, CL molecules appeared to escape the membrane together with the protein, which resulted in a loss of more than a half of CL content from the membrane. These findings may aid in understanding the early events leading to the remodeling of inner mitochondrial membrane and loss of its function during apoptosis.     

Expression of allograft inflammatory factor-1 (AIF-1) in response to bacterial challenge and tissue injury in the pearl oyster,Pinctada martensii

Allograft inflammatory factor-1 (AIF-1), an interferon (IFN)-γ-inducible calcium-binding cytokine, is associated with the inflammatory response and defense. We cloned and analyzed the expression pattern of the AIF-1 gene of the pearl oyster Pinctada martensii, hereafter designated PmAIF-1. The full-length PmAIF-1 cDNA is 946 bp in length and consists of a 5′-untranslated region (UTR) of 120 bp, a 3′-UTR of 376 bp, and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with an estimated molecular mass of 17 kDa. Sequence analysis reveals that PmAIF-1 contains two EF hand Ca⁺²-binding motifs like those in previously characterized AIF-1s while alignment with known AIF-1 protein sequences reveals higher similarity to invertebrate orthologs than to those of vertebrates.Quantitative PCR analysis reveals that PmAIF-1 is constitutively expressed, with the highest expression detected in hemocytes, and the expression level of PmAIF-1 mRNA was significantly up-regulated in hemocytes, gill, digestive gland under bacterial challenge and tissue injury. After challenged by gram-negative bacteria Vibrio alginolyticus and Vibrio parahaemolyticus, gram-positive bacteria Bacillus subtilis, the expression level of this gene in hemocytes were all up-regulated and reached the maximum point at 12 h (5.80 folds, P < 0.01), 6 h (5.02 folds, P < 0.01) and 12 h (5.49 folds, P < 0.01), respectively. Under shell damage and mantle injury, PmAIF-1 mRNA increased gradually in the first 3 h and reached a peak of expression at 6 h post-injury. These findings suggest that PmAIF-1 is an acute-response protein involved in the innate immune responses of pearl oysters, and provide general information about the mechanisms of innate immune defense against bacterial infection in pearl oysters.     

Syntheses,structures, and magnetic properties of heterobimetallic complexes based on tetracyanometallic building blocks

Two tetracyanometalate building blocks, [Fe(5,5′-dmbipy)(CN)₄]^? (2) and [Fe(4,4′-dmbipy)(CN)₄]^? (3) (5,5′-dmbipy = 5,5′-dimethyl-2,2′-bipyridine; 4,4′-dmbipy = 4,4′-dimethyl-2,2′-bipyridine), and two cyano-bridged heterobimetallic complexes, [Cu₂(bpca)₂(H₂O)₂Fe₂(5,5′-dmbipy)₂(CN)₈] · 2[Cu(bpca)Fe(5,5′-dmbipy)(CN)₄] · 4H₂O (4) and [Cu(bpca)Fe(4,4′-dmbipy)(CN)₄]_n (5) (bpca = bis(2-pyridylcarbonyl)amidate), have been synthesized and structurally characterized. Complex 4 contains two dinuclear and one tetranuclear heterobimetallic clusters in an asymmetric unit whereas the structure of complex 5 features a one-dimensional heterobimetallic zigzag chain. The Cu(II) ion is penta-coordinated in the form of a distorted square-based pyramid. Magnetic studies show ferromagnetic coupling between Cu(II) and Fe(III) ions with g = 2.28, J₁ = 2.64 cm^?1, J₂ = 5.40 cm^?1 and TIP = ?2.36 × 10^?3 for complex 4, and g = 2.17, J = 4.82 cm^?1 and zJ′ = 0.029 cm^?1 for complex 5.     

Syntheses and crystal structures of trimethyltin(IV) coordination polymers based on mixed ligands of 5-nitroisophthalate and 2,2′(4,4′)-bipy or phen

The trimethyltin(IV) polymer [(Me₃Sn)₂(nip) · EtOH]_n (1) of 5-nitroisophthalic acid (H₂nip) and its three derivatives with mixed organic N-donor ligands 2,2′-bipy [(Me₃Sn)₂(nip) · 2H₂O] · [(Me₃Sn)₂(nip) · H₂O] · 2(2,2′-bipy) (2) 4,4′-bipy {[(Me₃Sn)₂(nip)]₂(4,4′-bipy)}_n (3) or phen [(Me₃Sn)₂(nip) · H₂O] · (phen) (4) have been synthesized by the reaction of trimethyltin(IV) chloride and H₂nip when sodium ethoxide was added in the presence of 2,2′-bipy 4,4′-bipy or phen. All complexes 1–4 were characterized by elemental, IR, ¹H, ¹³C, and ¹¹⁹Sn NMR spectroscopy and X-ray crystallography analyses. Crystal, data collection and structure refinement parameters for complexes 1, 2, 3 and 4 are shown in Table 1, Table 2, respectively. The X-ray data showed the geometries of all the tin atoms in complexes 1–4 are trigonal bipyramidal. The X-ray analysis of 1 showed that the structure was a polymeric infinite chain with neighboring triorganotin centers being linked by dicarboxylate ligands and hydrogen bonds were found between adjacent chains. For 2, two different monomers were found, in one monomer, Me₃Sn were coordinated to both carboxyl groups of the ligand and water molecules were coordinated to the two Sn(IV) centers. In the other monomer, water molecules were coordinated to only one Sn center. Co-crystallized2,2′-bipy was found in 2 and a 2D supermolecular structure was formed via O–H⋯O and O–H⋯N (N atoms derived from 2,2′-bipy) hydrogen bonds. In 3 however, a 2D polymeric block was formed due to the inversion center of the 4,4′-bipy. For 4, due to the O–H⋯O and O–H⋯N (N atoms derived from phen) hydrogen bonds and intermolecular Sn⋯O bonds, a 2D polymeric structure was formed.     

Expanding the 4,4′-bipyridine ligand: Structural variation in {M(pytpy)2}2+ complexes (pytpy = 4′-(4-pyridyl)-2,2′:6′,2″-terpyridine,M = Fe,Ni, Ru) and assembly of the hydrogen-bonded,one-dimensional polymer {[Ru(pytpy)(Hpytpy)]}n3n+

The solid state structures of [Ni(1)₂][NO₃]₂ · 2MeOH · 2H₂O, [Fe(1)₂][ClO₄]₂ · 2MeOH · 0.5H₂O, [Ru(1)₂][PF₆]₂ and [Ru(1)₂][PF₆][NO₃] (1 = 4′-(4-pyridyl)-2,2′:6′,2″-terpyridine) are presented and the structural variation observed for the {M(1)₂}²⁺ unit is discussed. Protonation of the pendant pyridine group in [Ru(1)₂]²⁺ leads to the formation of a hydrogen-bonded, one-dimensional polymer [{Ru(1)(H1)}_n]³ⁿ⁺ exemplifed by the solid-state structure of [{Ru(1)(H1)}{Fe(NCS)₆} · 1.25H₂O]_n.     

Synthesis,crystal structure,and DNA-binding properties of a new copper (II) complex containing mixed-ligands of 2,2′-bipyridine and p-methylbenzoate

A novel copper complex of [Cu(bpy)(pba)₂ · H₂O] · 0.5H₂O (bpy = 2,2′-bipyridine, pba = p-methylbenzoate) was synthesized. The interaction of the complex to native fish sperm DNA was investigated through electrochemistry, electronic absorption spectroscopy and viscosity experiments. In the X-ray crystallography structure, the copper (II) ion is coordinated by two oxygen atoms of two p-methylbenzoate groups, two nitrogen atoms of 2,2′-bipyridine and one water molecule. The observed changes in the physicochemical features of the copper (II) complex on binding to DNA suggested that the complex bind to DNA with intercalation mode via 2,2′-bipyridine ring into DNA base pairs. Electrochemical studies revealed that the complex prefer to bind to DNA in Cu(I) form rather than Cu(II) oxidation state form. Additionally, the nuclease activity of the title complex was assessed by gel electrophoresis assay and the results shown that the copper complex can cleave pBR322 DNA effectively in the presence of ascorbic acid.     

The 2-D double layer structures of indium–benzenemulticarboxylate coordination polymers with green fluorescence

Two coordination polymers, [In(btc)(2,2′-bpy)(H₂O)]_n · nH₂O (1) and [In₂(btec)(2,2′-bpy)₂Cl₂]_n (2) (H₃btc = 1,3,5-benzenetricarboxylic acid, H₄btec = 1,2,4,5-benzenetetracarboxylic acid, 2,2′-bpy = 2,2′-bipyridine), have been prepared under hydrothermal conditions. Single-crystal X-ray diffraction analyses reveal that compounds 1 and 2 consist of two-dimensional double layer structures with (3, 3) grids and (4, 2) grids, respectively. The grids along two different directions in 1 are of the opposite chirality, while the interactions of double-layer sheets in 2 create a three-dimensional supramolecular network with one-dimensional tunnels. The additional green luminescences for both 1 and 2 in the solid state at low temperature imply the red shift of emission energy in the compounds, which may derive from LMCT. Their X-ray powder diffractions and thermogravimetric analyses are also discussed.     

A 5'-flanking region of embryonic-type myosin heavy chain gene,MYHM743-2, from torafugu Takifugu rubripes regulates developmental muscle-specific expression

The myosin heavy chain gene, MYH_M743-2, is highly expressed in fast muscle fibers of torafugu embryos. However, the regulatory mechanisms involved in its expression have been unclear. In this study, we examined spatio-temporal expression patterns of this gene during development by injecting expression vectors containing the GFP reporter gene fused to the 5'-flanking region of MYH_M743-2 into fertilized eggs of zebrafish and medaka. Although the -2.1 kb 5'-flanking region of torafugu MYH_M743-2 showed no homology with the corresponding regions of zebrafish and medaka orthologous genes on the rVISTA analysis, the torafugu 5'-flanking region activated the GFP expression which was detected in the myotomal compartment for both zebrafish and medaka embryos. The GFP expression was localized to fast and slow muscle fibers in larvae as revealed by immunohistochemical analysis. In addition to the above tissues, GFP was also expressed in jaw, eye and pectoral fin muscles in embryos and larvae. These results clearly demonstrated that the 2.1 kb 5'-flanking region of MYH_M743-2 contains essential cis-regulatory sequences for myogenesis that are conserved among torafugu, zebrafish and medaka.     

Design,synthesis, and DNA binding characteristics of a group of orthogonally positioned diamino,N-formamido,pyrrole- and imidazole-containing polyamides

Orthogonally positioned diamino/dicationic polyamides (PAs) have good water solubility and enhanced binding affinity, whilst retaining DNA minor groove and sequence specificity compared to their monoamino/monocationic counterparts. The synthesis and DNA binding properties of the following diamino PAs: f-IPI (3a), f-IPP (4), f-PIP (5), and f-PPP (6) are described. P denotes the site where a 1-propylamino group is attached to the N1-position of the heterocycle. Binding of the diamino PAs to DNA was assessed by DNase I footprinting, thermal denaturation, circular dichroism titration, biosensor surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) studies. According to SPR studies, f-IPI (3a) bound more strongly (K_eq = 2.4 × 10⁸ M^?1) and with comparable sequence selectivity to its cognate sequence 5′-ACGCGT-3′ when compared to its monoamino analog f-IPI (1). The binding of f-IPI (3a) to 5′-ACGCGT-3′ via the stacked dimer motif was balanced between enthalpy and entropy, and that was quite different from the enthalpy-driven binding of its monoamino parent f-IPI (1). f-IPP (4) also bound more strongly to its cognate sequence 5′-ATGCAT-3′ (K_eq = 7.4 × 10⁶ M^?1) via the side-by-side stacked motif than its monoamino analog f-IPP (2a). Although f-PPP (6) bound via a 1:1 motif, it bound strongly to its cognate sequence 5′-AAATTT-3′ (K_eq = 4.8 × 10⁷ M^?1), 15-times higher than the binding of its monoamino analog f-PPP (2c), albeit f-PPP bound via the stacked motif. Finally, f-PIP (5) bound to its target sequence 5′-ATCGAT-3′ as a stacked dimer and it has the lowest affinity among the diamino PAs tested (K_eq <1 × 10⁵ M^?1). This was about two times lower in affinity than the binding of its monoamino analog f-PIP (2b). The results further demonstrated that the ‘core rules’ of DNA recognition by monoamino PAs also apply to their diamino analogs. Specifically, PAs that contain a stacked IP core structure bind most strongly (highest binding constants) to their cognate GC doublet, followed by the binding of PAs with a stacked PP structure to two degenerate AT base pairs, and finally the binding of PAs with a PI core to their cognate CG doublet.     

3′-Oxo-, amino-, thio- and sulfone-acetic acid modified thymidines: Effect of increased acidity on ribonuclease A inhibition

A family of 3′-functionalized thymidines carrying XCH₂COOH (X = O, NH, S, SO₂) groups has been designed as inhibitors of RNase A. This is because it is possible to manipulate the overall acidity of this new class of nucleic ‘acids’ by changing X from oxygen to the SO₂ group in the series. It is also expected that the acyclic nature of the XCH₂COOH group would provide enough flexibility to the –COOH group to have maximum interactions with the catalytic subsite P₁ of RNase A. As the –SO₂CH₂COOH substituted derivative showed better potency partially because of the increased acidity of the –COOH group, the inhibitory properties of both 5′-substituted and 3′,5′-disubstituted sulfone acetic acid modified thymidines were investigated. Two –SO₂CH₂COOH groups were incorporated with the expectation of targeting two phosphate binding sites simultaneously. Thus, 3′,5′-dideoxy-3′,5′-bis-S-[(carboxymethyl)sulfonyl]thymidine emerged as the best inhibitor in this series with a K_i value of 25 ± 2 μM.     

Synthesis and characterization of ampelopsin glucosides using dextransucrase from Leuconostoc mesenteroides B-1299CB4: Glucosylation enhancing physicochemical properties

Novel ampelopsin glucosides (AMPLS-Gs) were enzymatically synthesized and purified using a Sephadex LH-20 column. Each structure of the purified AMPLS-Gs was determined by nuclear magnetic resonance, and the ionic product of AMPLS-G1 was observed at m/z 505 (C₂₁H₂₂O₁₃·Na)⁺ using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. AMPLS-G1 was identified as ampelopsin-4′-O-α-d-glucopyranoside. The optimum condition for AMPLS-G1, determined using response surface methodology, was 70 mM ampelopsin, 150 mM sucrose, and 1 U/mL dextransucrase, which resulted in an AMPLS-G1 yield of 34 g/L. The purified AMPLS-G1 displayed 89-fold increased water solubility and 14.5-fold browning resistance compared to those of AMPLS and competitive inhibition against tyrosinase with a K_i value of 40.16 μM. This value was smaller than that of AMPLS (K_i = 62.56 μM) and much smaller than that of β-arbutin (K_i = 514.84 μM), a commercial active ingredient of whitening cosmetics. These results indicate the potential of AMPLS and AMPLS-G1 as superior ingredients for functional cosmetics.     

Characterization of bi-functional CYP154 from Nocardia farcinica IFM10152 in the O-dealkylation and ortho-hydroxylation of formononetin

Among the 27 cytochrome P450s (CYPs) of Nocardia farcinica IFM10152, three CYPs have been identified as having O-dealkylation catalytic activity. Of the two that encode CYP154 subfamilies, the one encoded by the nfa22930 gene showed distinct O-dealkylation and subsequent hydroxylation of formononetin. Firstly, formononetin was O-dealkylated into daidzein, which was subsequently mono-hydroxylated at the 3′-position of the B-ring into ortho-dihydroxy-isoflavone. Apparent k_cat/K_m values of CYP154 for the O-dealkylation of formononetin and the hydroxylation of daidzein were 3.57 and 1.84 μM⁻¹ min⁻¹, respectively. The dissociation constants of CYP154 based on spectral changes upon binding to each substrate were 5.16 and 3.11 μM, respectively. Homology modeling and docking simulation found that Thr247 is responsible for the 3′-position hydroxylation reaction by forming a hydrogen bond with the 4′-hydroxyl group of daidzein that forces the proton at the 3′-position to face the heme center. Site-directed mutagenesis of Thr247 to alanine drastically decreased the binding affinity for daidzein (9.73 μM) as well as 3′-position hydroxylation catalytic activity by 3 fold (0.48 μM⁻¹ min⁻¹).     

Hydrothermal syntheses and characterizations of two novel heteropolytungstate supported coordination compounds

Two novel heteropolytungstates, [Ni(2,2′-bpy)₃]_1.5{PW_10.79V_1.21O₄₀(VO)_0.5[Ni(2,2′-bpy)₂L]} · 0.5H₂O (1) (2,2′-bpy = 2,2′-bipyridine, L = 0.5H₂O + 0.5OH^?) and K[PW₁₂O₄₀[Ni(1,10-phen)₂(OH)]₂] · 2H₂O (2) (1,10-phen = 1,10-phenanthroline), have been synthesized under hydrothermal conditions and characterized by IR, XPS, and X-ray diffraction analyses. The polyoxoanion of 1 is a mono-capped α-Keggin cluster which supports a metal coordination fragment [Ni(2,2′-bpy)₂L]^1.5+, while the structure of 2 exhibits a 1D chain constructed from pseudo-Keggin cluster bi-supported transition metal complexes linked by K⁺ ions. Studies of magnetic properties indicated the presence of paramagnetic behaviours for compounds 1 and 2.