Comparative homology modeling-inspired protein engineering for improvement of catalytic activity of Mugil cephalus epoxide hydrolase

The epoxide hydrolase (EH) of a marine fish, Mugil cephalus, was engineered to improve the catalytic activity based on comparative homology modeling. The 3-D crystal structure of the EH from Aspergillus niger was used as a template. A triple point mutant, F193Y for spatial orientation of the nucleophile (D199), W200L for removing electron density overlap between W200 and Y348, and E378D for good charge relay in the active site, was developed. The initial hydrolysis rate, the reaction time to reach 98 %ee, and yield were enhanced up to 35-fold, 26-fold and 32%, respectively, by homology modeling-inspired site-directed mutagenesis of M. cephalus EH.     

Enhancement of Riptortus clavatus (Thunberg) (Hemiptera: Alydidae) egg parasitism by a component of the bug's aggregation pheromone

Egg parasitism of the bean bug, Riptortus clavatus (Thunberg) (Hemiptera: Alydidae), was surveyed using individual egg bags in a sweet persimmon orchard and at Gyeongsang National University (GNU) campus, Korea in 2006. The effect of (E)-2-hexenyl (Z)-3-hexenoate (E2HZ3H), one component of R. clavatus aggregation pheromone, on the parasitism enhancement was tested at GNU campus and in a soybean field in 2005 and 2006, respectively. In 2006, two egg parasitoid species, Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae) and Gryon japonicum (Ashmead) (Hymenoptera: Scelionidae), emerged from R. clavatus eggs. Parasitism by O. nezarae was 9.4–48.3% in mid August to mid September in GNU campus and 6.7–30.0% at the orchard. Total parasitism by G. japonicum (2.5%) at both sites throughout the experimental period was lower than that by O. nezarae (12.5%). This survey revealed nearly no activity of the two species after October at both sites. G. japonicum was solitary and O. nezarae could be either solitary or gregarious. From a single R. clavatus egg, one female or one male G. japonicum emerged. However, an average of 4.3 O. nezarae emerged from one host egg. It took 12.6 d for G. japonicum and 12.0 d for O. nezarae to emerge from R. clavatus eggs in the laboratory. Treatment with E2HZ3H increased parasitism by O. nezarae in both years, but did not increase parasitism by G. japonicum. This suggests that E2HZ3H can be used as a kairomone to reduce the density of R. clavatus in the fields where natural parasitism by O. nezarae is high.     

Molecular Cloning of Cyanobacterial Pteridine Glycosyltransferases That Catalyze the Transfer of either Glucose or Xylose to Tetrahydrobiopterin

Here, we report cloning of cyanobacterial genes encoding pteridine glycosyltransferases that catalyze glucosyl or xylosyl transfer from UDP-sugars to tetrahydrobiopterin. The genes were cloned by PCR amplification from genomic DNA which was isolated from culture and environmental samples and overexpressed in Escherichia coli for an in vitro activity assay.Tetrahydrobiopterin (BH4) is well known among pteridine compounds as a cofactor for aromatic amino acid hydroxylases and nitric oxide synthases in animals (19). Pteridine glycosides such as biopterin and 6-hydroxymethylpterin glycosides have been found in cyanobacteria and anaerobic photosynthetic bacteria (2, 4, 5, 8, 11, 13, 15, 17, 21). Although the function of these glycosides remains unknown, they are abundant and ubiquitous in cyanobacteria, implying some essential role (3, 6, 16-18, 21). There is a group of enzymes, named pteridine glycosyltransferases (PGTs), known to catalyze a variety of glycosyl transfers to pteridines. The first PGT isolated from the cyanobacterium Synechococcus sp. PCC 7942 was shown to catalyze a glucosyl transfer from UDP-glucose to BH4 and was therefore named UDP-glucose:BH4 glucosyltransferase (BGluT) (7). After cloning of the gene encoding BGluT (6), a PGT that catalyzes the transfer of glucuronic acid for cyanopterin synthesis was identified (12). In addition, there are many putative PGT homologs encoded in bacterial genomes, although their exact catalytic functions have not been determined. We recently found that BGluT is useful for the simultaneous detection of oxidized and reduced forms of BH4 in animal samples (14). Glycosyltransferases are also being studied intensively for applications in the design of novel pharmaceutical derivatives (1, 10). We were thus encouraged to find PGTs with new substrate specificities or enzymatic properties not only for study of protein structure and function but also for application in BH4 research. In this study, we succeeded in cloning four cyanobacterial genes encoding PGTs with either glucosyl- or xylosyltransferase activity, and here we report the results.PGT genes were cloned from Arthrospira platensis CY-007 (obtained through Hawaii Oceanic Institute sampling) and Arthrospira maxima CY-049 (UTEX 2342), which were cultured in the Korea Marine Microalgae Culture Center, and from environmental DNA sequences (designated UCNR-001 and UCNR-002) isolated from wild algal mats in the Nakdong River, South Korea. In order to amplify conserved internal sequences of the unknown PGT genes, degenerate PCR primers were designed from the nucleotide sequences of cyanobacterial PGT homologs using GeneFisher2 (9). A protein homology search with BGluT against the bacterial genome database in NCBI revealed more than a hundred PGT homologs. When a phylogenetic tree was constructed from the putative sequences, there was a separate group comprising cyanobacterial PGTs. Figure ​Figure11 shows the cyanobacterial cluster, in which members shared sequence identities of more than 34%. Because the degenerate primers designed from all of the cyanobacterial PGTs were too highly degenerate, the cluster was divided into four subgroups, as shown in Fig. ​Fig.1:1: this division allowed primers to be designed for each of the four subgroups. The PGTs in subgroup I were clearly distinguishable from the others, because they all originated from marine picocyanobacteria, which are abundant in the pelagic realm. Subgroup I could be divided further into two groups comprising PGTs from either Prochlorococcus species (CIA) or marine Synechococcus species (CIB). Subgroup II was also divided into two groups, CIIA, consisting mostly of PGTs from Synechococcus species, and CIIB, containing the other PGTs. Among the primers designed for each subgroup, those for the CIIA and CIIB subgroups successfully amplified DNA sequences of the expected sizes. The primer sequences were 5′-GTTCAGGAWTAGGAGGTGGAGT-3′ (CIIA-forward)/5′-CGCYTCAATWGCTACATTTCCA-3′ (CIIA-reverse) and 5′-ACGACTGGCTMYCGYTTTAYCTGA-3′ (CIIB-forward)/5′-GCYTCCACCCAYTTRGGGGTCA-3′ (CIIB-reverse). Based on the determined partial gene sequences, additional sets of primer pairs were designed for the inverse PCR method (20). The sequences were 5′-GATGAACTACAACAGGGTCTGCGTC-3′ (CY-007 forward)/5′-CGGCTTTTTAAGGCTTTTGCCATATTC-3′ (CY-007 reverse), 5′-GTCTGCGTGAATGTCGAGG-3′ (CY-047 forward)/5′-ATGACCTCGGCTGTGTAAG-3′ (CY-047 reverse), and 5′-CCTACAAAAAGAGCTAGGCGACTGTTTTG-3′ (UCNR forward)/5′-CCAAAGAAACGGAAGCCATGCTG-3′ (UCNR reverse). Total genomic DNA samples were partially digested with RsaI and then self-ligated to be used as templates for PCR amplification with the primer pairs. The amplified DNA sequences revealed the missing 5′- and 3′-end sequences of the genes.Open in a separate windowFIG. 1.Neighbor-joining phylogenetic tree of cyanobacterial PGT protein sequences, identified by NCBI accession numbers. Bootstrap values are presented at the nodes. The names of strains whose PGTs are characterized are in bold.The deduced protein sequences were multiply aligned with BGluT (Fig. ​(Fig.2).2). Amino acid identities for all sequences in pairwise comparisons are given as percentages in Fig. ​Fig.2.2. Recently, draft assemblies of the genome sequences of Arthrospira platensis strain Paraca and Arthrospira maxima CS-328 (UTEX 2342) were announced. The annotated PGT (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"EDZ91868","term_id":"209491491","term_text":"EDZ91868"}}EDZ91868) of Arthrospira maxima CS-328 was identical to the PGT of CY-049 at both the amino acid and nucleotide levels, proving that the two organisms originated from the same UTEX stock (UTEX 2342). On the other hand, the PGTs of Arthrospira platensis strains Paraca and CY-007 were different at nine individual nucleotides, resulting in seven amino acid differences. A phylogenetic analysis showed that CY-007 and CY-049 PGTs belonged to the CIIB subgroup and that UCNR-001 and UCNR-002 PGTs clustered in the CIIA subgroup (data not shown).Open in a separate windowFIG. 2.Alignment of multiple PGT sequences. Conserved sequences are shaded at four levels using GeneDoc software. At the end of the alignment, amino acid identities in percentages are given for all sequences in pairwise comparisons.In order to identify the catalytic function of the putative PGTs, the recombinant proteins were produced in Escherichia coli. The complete open reading frame (ORF) sequences were amplified by PCR from the genomic DNA samples, cloned into the pGEM-T vector, and subsequently cloned as NdeI/BamHI restriction fragments into pET-28b (for CY-007 and CY-049 sequences) or pET-15b (for UCNR-001 and UCNR-002 sequences). E. coli BL21(DE3)/pLysS transformants were induced with 0.05 to 0.2 mM isopropyl-β-d-thiogalactopyranoside and were cultured for 8 h at 22°C. The recombinant proteins were purified by chromatography on Ni-nitrilotriacetic acid gel according to the instructions of the manufacturer (Qiagen). The proteins were eluted with 250 mM imidazole, dialyzed against a mixture of 20 mM Tris-HCl (pH 7.5) and 30% (vol/vol) glycerol, and stored in aliquots at −70°C until use. Purification of the proteins was confirmed by electrophoresis on an SDS-polyacrylamide gel (Fig. ​(Fig.3A).3A). BGluT from a previous purification was used (6). Aliquots of PGT were assayed at 37°C for 10 min in a reaction mixture of 100 μl containing 50 mM sodium phosphate, pH 7.5, 10 mM MnCl₂, 0.2% ascorbic acid, 1 μM BH4 (Schircks Lab, Switzerland), and 100 μM UDP-glucose or UDP-xylose. The reaction mixture was combined with an equal volume of acidic iodine solution (2% KI and 1% I₂ in 1 N HCl) for 1 h in the dark. After centrifugation, the supernatant was mixed in a 10:1 volume ratio with 5% ascorbic acid and subjected to high-performance liquid chromatography (HPLC). HPLC was performed with a Gilson 321 pump equipped with an Inertsil ODS-3 column (150 by 2.3 mm; particle size, 5 μm [GL Science, Japan]) and a fluorescence detector (Shimadzu RF-10AXL). Pteridines were eluted with 10 mM potassium phosphate buffer (pH 6.0) at a flow rate of 1.2 ml/min and were monitored at excitation and emission wavelengths of 350 and 450 nm, respectively.Open in a separate windowFIG. 3.Analysis of purified recombinant PGTs on an SDS-12.5% polyacrylamide gel (A) and HPLC analysis of the enzymatic products (B).The enzymatic products of PGTs (Fig. ​(Fig.3)3) appeared only when enzymes were incubated with BH4 as a sugar acceptor and either UDP-glucose (for CY-007, UCNR-001, and UNCR-002 PGTs) or UDP-xylose (for CY-049 PGT) as a sugar donor. HPLC analysis of cultured CY-007 and CY-049 cells confirmed the presence of the corresponding biopterin glycosides (data not shown), supporting the conclusion that the PGTs exhibited genuine in vivo activities. This is the first report of a gene encoding a PGT that catalyzes xylosyl transfer to BH4. Although the data are not shown here, we found additional xylosyl transfer PGTs in Anabaena sp. PCC 7120, Gloeobacter violaceus PCC 7421, and Thermosynechococcus elongatus BP-1, whose genomic sequences were determined. The putative PGT genes (represented in Fig. ​Fig.1)1) were amplified by PCR from the genomic DNA, which was a kind gift from the Kazusa DNA Research Institute (http://genome.kazusa.or.jp/cyanobase/). The recombinant proteins for the in vitro activity assay were prepared by cloning the genes into pET-28b and overexpressing the proteins in E. coli according to the same procedures performed for the other PGTs. Interestingly, CY-007 and CY-049 PGTs exhibited different substrate specificities, although they share 93% protein sequence identity, and they also had higher specific activities than the other PGTs (Fig. ​(Fig.4).4). The three-dimensional structures of the proteins are currently being investigated to further understanding of the structural properties involved. Considering the cyanobacterial PGTs hitherto identified, there seems to be little correlation between their substrate preferences and phylogenetic classification. However, the CI group PGTs, which diverged early from the CII group PGTs, might have some distinctive features. Finally, the successful cloning of PGT genes from environmental DNA allows for potentially new PGTs to be isolated from cyanobacteria, which are abundant in nature.Open in a separate windowFIG. 4.Comparative analysis of PGT activities. The maximal activity (100%) corresponds to complete glycosylation of 1 μM BH4 in the reaction mixture, which contained 0.5 mM UDP-xylose for CY-049 PGT or 0.5 mM UDP-glucose for the other PGTs. The mixtures were incubated for 10 min with the indicated amounts of proteins.     

Crystal structure of the MukB hinge domain with coiled‐coil stretches and its functional implications

The structural maintenance of chromosomes (SMC) family proteins are commonly found in the multiprotein complexes involved in chromosome organization, including chromosome condensation and sister chromatid cohesion. These proteins are characterized by forming a V‐shaped homo‐ or heterodimeric structure with two long coiled‐coil arms having two ATPase head domains at the distal ends. The hinge domain, located in the middle of the coiled coil, forms the dimer interface. In addition to being the dimerization module, SMC hinges appear to play other roles, including the gateway function for DNA entry into the cohesin complex. Herein, we report the homodimeric structure of the hinge domain of Escherichia coli MukB, which forms a prokaryotic condensin complex with two non‐SMC subunits, MukE and MukF. In contrast with SMC hinge of Thermotoga maritima which has a sizable central hole at the dimer interface, MukB hinge forms a constricted dimer interface lacking a hole. Under our assay conditions, MukB hinge does not interact with DNA in accordance with the absence of a notable positively charged surface patch. The function of MukB hinge appears to be limited to dimerization of two copies of MukB molecules. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Role of Pin1 in neointima formation: Down-regulation of Nrf2-dependent heme oxygenase-1 expression by Pin1

Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to intima formation after stenting and balloon angioplasty. Pin1, a peptidyl prolyl isomerase recognizing phosphorylated Ser/Thr–Pro, isomerizes the peptide bond. Because Pin1 overexpression is associated with transformation and the uncontrolled cell growth of tumors, we hypothesized that Pin1 functions as a chronic stimulator of VSMC proliferation. Pin1-positive smooth muscle cells were seen in the neointimal region of the femoral artery after guidewire injury. Exposure of VSMCS to platelet-derived growth factor (PDGF) increased Pin1 expression in a concentration-dependent manner. Basal cell growth rate and cyclin D1 expression were enhanced in Pin1-overexpressing VSMCs (Pin1-VSMCs). Moreover, PDGF-induced production of reactive oxygen species (ROS) in Pin1-VSMCs was higher than in control VSMCs. In Pin1-VSMCs, heme oxygenase-1 (HO-1) induction in response to nitric oxide donor was suppressed compared to control VSMCs. Nuclear translocation of nuclear factor E2-related factor-2 (Nrf2) was also diminished in Pin1-VSMCs. In contrast, the activity of the inducible minimal antioxidant response element (ARE) was potentiated in Pin1-null mouse embryonic fibroblasts (MEFs), compared to Pin1-wild-type MEFs. Moreover, Nrf2 ubiquitination was stimulated by Pin1 overexpression. Intraperitoneal injection of juglone (a Pin1 inhibitor) for 3 weeks (1 mg/kg, two times a week) significantly suppressed neointimal formation induced by wire injury. In conclusion, Pin1 induction during neointimal formation may be associated with ROS-mediated VSMC proliferation via down-regulation of Nrf2/ARE-dependent HO-1 expression. Pin1 may be a novel therapeutic target for several vascular diseases including atherosclerosis and stenosis.     

Arabidopsis SCP1-like small phosphatases differentially dephosphorylate RNA polymerase II C-terminal domain

RNA polymerase II carboxyl-terminal domain (pol II CTD) phosphatases that can dephosphorylate both Ser2-PO₄ and Ser5-PO₄ of CTD have been identified in animals and yeasts, however, only Ser5-PO₄-specific CTD phosphatases have been identified in plants. Among predicted Arabidopsis SCP1-like small phosphatases (SSP), SSP4, SSP4b, and SSP5 form a unique group with long N-terminal extensions. While SSPs’ expression showed similar tissue-specificities, SSP4 and SSP4b were localized exclusively in the nuclei, whereas SSP5 accumulated in both nuclei and cytoplasm. Detailed characterization of SSP activities using various peptides and full-length Arabidopsis pol II CTD substrates established that SSP4 and SSP4b could dephosphorylate both Ser2-PO₄ and Ser5-PO₄ of CTD, whereas SSP5 dephosphorylated only Ser5-PO₄. These results indicate that Arabidopsis SSP gene family encodes active CTD phosphatases like animal SCP1 family proteins, with distinct substrate specificities.     

Mitogen‐activated protein kinase 3 and 6 regulate Botrytis cinerea‐induced ethylene production in Arabidopsis

Plants challenged by pathogens, especially necrotrophic fungi such as Botrytis cinerea, produce high levels of ethylene. At present, the signaling pathways underlying the induction of ethylene after pathogen infection are largely unknown. MPK6, an Arabidopsis stress‐responsive mitogen‐activated protein kinase (MAPK) was previously shown to regulate the stability of ACS2 and ACS6, two type I ACS isozymes (1‐amino‐cyclopropane‐1‐carboxylic acid synthase). Phosphorylation of ACS2 and ACS6 by MPK6 prevents rapid degradation of ACS2/ACS6 by the 26S proteasome pathway, resulting in an increase in cellular ACS activity and ethylene biosynthesis. Here, we show that MPK3, which shares high homology and common upstream MAPK kinases with MPK6, is also capable of phosphorylating ACS2 and ACS6. In the mpk3 mutant background, ethylene production in gain‐of‐function GVG‐NtMEK2^DD transgenic plants was compromised, suggesting that MPK6 and MPK3 function together to stabilize ACS2 and ACS6. Using a liquid‐cultured seedling system, we found that B. cinerea‐induced ethylene biosynthesis was greatly compromised in mpk3/mpk6 double mutant seedlings. In contrast, ethylene production decreased only slightly in the mpk6 single mutant and not at all in the mpk3 single mutant, demonstrating overlapping roles for these two highly homologous MAPKs in pathogen‐induced ethylene induction. Consistent with the role of MPK3/MPK6 in the process, mutation of ACS2 and ACS6, two genes encoding downstream substrates of MPK3/MPK6, also reduced B. cinerea‐induced ethylene production. The residual levels of ethylene induction in the acs2/acs6 double mutant suggest the involvement of additional ACS isoforms, possibly regulated by MAPK‐independent pathway(s).