Cloning, Baeyer-Villiger biooxidations, and structures of the camphor pathway 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetyl-coenzyme A monooxygenase of Pseudomonas putida ATCC 17453

A dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ³-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor by Pseudomonas putida ATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ³-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) in Escherichia coli and determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP⁺ at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP⁺. A comparison of several crystal forms of OTEMO bound to FAD and NADP⁺ revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ³-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (k_cat/K_m) favors 2-n-hexyl cyclopentanone (4.3 × 10⁵ M⁻¹ s⁻¹) as a substrate, although its affinity (K_m = 32 μM) was lower than that of the CoA-activated substrate (K_m = 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.     

Evidence for a dual role of an active site histidine in α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase

The previously reported crystal structures of α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) show a five-coordinate Zn(II)(His)(3)(Asp)(OH(2)) active site. The water ligand is H-bonded to a conserved His228 residue adjacent to the metal center in ACMSD from Pseudomonas fluorescens (PfACMSD). Site-directed mutagenesis of His228 to tyrosine and glycine in this study results in a complete or significant loss of activity. Metal analysis shows that H228Y and H228G contain iron rather than zinc, indicating that this residue plays a role in the metal selectivity of the protein. As-isolated H228Y displays a blue color, which is not seen in wild-type ACMSD. Quinone staining and resonance Raman analyses indicate that the blue color originates from Fe(III)-tyrosinate ligand-to-metal charge transfer. Co(II)-substituted H228Y ACMSD is brown in color and exhibits an electron paramagnetic resonance spectrum showing a high-spin Co(II) center with a well-resolved (59)Co (I = 7/2) eight-line hyperfine splitting pattern. The X-ray crystal structures of as-isolated Fe-H228Y (2.8 ?) and Co-substituted (2.4 ?) and Zn-substituted H228Y (2.0 ? resolution) support the spectroscopic assignment of metal ligation of the Tyr228 residue. The crystal structure of Zn-H228G (2.6 ?) was also determined. These four structures show that the water ligand present in WT Zn-ACMSD is either missing (Fe-H228Y, Co-H228Y, and Zn-H228G) or disrupted (Zn-H228Y) in response to the His228 mutation. Together, these results highlight the importance of His228 for PfACMSD's metal specificity as well as maintaining a water molecule as a ligand of the metal center. His228 is thus proposed to play a role in activating the metal-bound water ligand for subsequent nucleophilic attack on the substrate.     

Large-scale development of expressed sequence tag-derived simple sequence repeat markers and diversity analysis in Arachis spp.

Large-scale development of expressed sequence tag simple sequence repeat (EST-SSR) markers was performed in peanut (Arachis hypogaea L.) to obtain more informative genetic markers. A total of 10,102 potential non-redundant EST sequences, including 3,445 contigs and 6,657 singletons, were generated from cDNA libraries of the gynophore, roots, leaves and seedlings. A total of 3,187 primer pairs were designed on flanking regions of SSRs, some of which allowed one and two base mismatches. Among the 3,187 markers generated, 2,540 (80%) were trinucleotide repeats, 302 (9%) were dinucleotide repeats, and 345 (11%) were tetranucleotide repeats. Pre-polymorphic analyses of 24 Arachis accessions were performed using 10% polyacrylamide gels. A total of 1,571 EST-SSR markers showing clear polymorphisms were selected for further polymorphic analysis with a Fluoro-fragment Analyzer. The 16 Arachis accessions examined included cultivated peanut varieties as well as diploid species with the A or B genome. Altogether 1,281 (81.5%) of the 1,571 markers were polymorphic among the 16 accessions, and 366 (23.3%) were polymorphic among the 12 cultivated varieties. Diversity analysis was performed and the genotypes of all 16 Arachis accessions showed similarity coefficients ranging from 0.37 to 0.97. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9604-8) contains supplementary material, which is available to authorized users.     

Tissue localization of maize acetylcholinesterase associated with heat tolerance in plants

Our recent study reported that maize acetylcholinesterase (AChE) activity in the coleoptile node is enhanced through a post-translational modification response to heat stress and transgenic plants overexpressing maize AChE gene had an elevated heat tolerance, which strongly suggests that maize AChE plays a positive, important role in maize heat tolerance. Here we present (1) maize AChE activity in the mesocotyl also enhances during heat stress and (2) maize AChE mainly localizes in vascular bundles including endodermis and epidermis in coleoptile nodes and mesocotyls of maize seedlings.     

The CCR4-NOT complex is implicated in the viability of aneuploid yeasts

To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast Schizosaccharomyces pombe, in which most types of aneuploidy are eventually lethal to the cell. Aneuploids remain viable for a period of time and can form colonies by reducing the extent of the aneuploidy. We hypothesized that a reduction in colony formation efficiency could be used to screen for gene deletions that compromise aneuploid viability. Deletion mutants were used to measure the effects on the viability of spores derived from triploid meiosis and from a chromosome instability mutant. We found that the CCR4-NOT complex, an evolutionarily conserved general regulator of mRNA turnover, and other related factors, including poly(A)-specific nuclease for mRNA decay, are involved in aneuploid viability. Defective mutations in CCR4-NOT complex components in the distantly related yeast Saccharomyces cerevisiae also affected the viability of spores produced from triploid cells, suggesting that this complex has a conserved role in aneuploids. In addition, our findings suggest that the genes required for homologous recombination repair are important for aneuploid viability.     

Pirfenidone inhibits the expression of HSP47 in TGF-beta1-stimulated human lung fibroblasts

Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone) is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and patients with idiopathic pulmonary fibrosis (IPF). Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen and plays an important role in the pathogenesis of IPF. The present study evaluated the in vitro effects of pirfenidone on expression of HSP47 and collagen type I in cultured normal human lung fibroblasts (NHLF). Expression levels of HSP47 and collagen type I in NHLF stimulated by transforming growth factor (TGF)-beta1 were evaluated genetically, immunologically and immunocytochemically. Treatment with TGF-beta1 stimulated both mRNA and protein expressions of both HSP47 and collagen type I in NHLF, and pirfenidone significantly inhibited this TGF-beta1-enhanced expression in a dose-dependent manner. We concluded that the anti-fibrotic effect of pirfenidone may be mediated not only through direct inhibition of collagen type I expression but also at least partly through inhibition of HSP47 expression in lung fibroblasts, with a resultant reduction of collagen synthesis in lung fibrosis.     

Cloning of the pyridoxine 5'-phosphate phosphatase gene (pdxP) and vitamin B6 production in pdxP recombinant Sinorhizobium meliloti

A novel gene (pdxP) encoding a pyridoxine 5'-phosphate (PNP) phosphatase involved in the last step of pyridoxine biosynthesis was cloned from Sinorhizobium meliloti IFO 14782 on the basis of the peptide sequences of the natural enzyme. The pdxP gene is an open reading frame (708 bp) encoding 235 amino acid residues with a calculated molecular weight of 26,466. From its deduced amino acid sequence, it was predicted that the enzyme belongs to the haloacid dehalogenase superfamily. Transformants of Escherichia coli and S. meliloti by pdxP gene expression plasmids showed stimulated PNP phosphatase activities. When pdxP was overexpressed together with the PNP synthase gene (pdxJ) in S. meliloti, the recombinant strain produced 149 mg/l of pyridoxine, 46% and 16% higher than the host strain and the pdxJ recombinant of S. meliloti respectively.     

Fundal height as a predictor of early preterm triplet delivery.

The purpose of this study was to create graphs of fundal height parameters in triplet pregnancies compared with those in twin pregnancies, and to investigate whether larger fundal heights in triplet pregnancies would predispose them to earlier delivery (before 34 weeks). The subjects were 727 twin pregnant women and 133 triplet pregnant women, who delivered after 1984. Triplet pregnancies showed significantly higher fundal heights compared with twin pregnancies at each gestational age (weeks). In triplet pregnancies, a single fundal height measurement above the 90th percentile before 34 weeks yielded a sensitivity of 31.3% and specificity of 82.4% for delivery before 34 weeks, with a positive predictive value of 50.0% and a negative predictive value of 68.0%. After adjusting for each associated factor using logistic regression, the risk of preterm labour was not significantly associated with a single fundal height measurement above the 90th percentile recorded before 34 weeks.     

Seasonal Changes in Nitrogen Fractions of Coptis japonica, a Temperate Forest Evergreen Chamaephyte, and their Ecological Implications

Seasonal changes in several forms of nitrogen were investigatedin Coptis japonica, an evergreen rosette hemicryptophyte intemperate deciduous forest. The concentration of total nitrogenin rhizomes and roots decreased during the period of new shootgrowth from winter to spring. In the rhizomes, total solubleprotein stored by early summer decreased gradually until winter,coupled with an increase in free amino acids. Nitrogen was largelystored in free amino acids in the roots, especially during summer.The total soluble protein in current-year leaves decreased fromspring to summer and then increased during winter. The seasonalchanges in nitrogen components were coincident with the changein light-saturated photosynthetic rates recorded in a previousstudy. The ratio of total soluble protein to total nitrogendecreased from spring to summer and then increased from latesummer to winter in the current-year leaves. In contrast, chlorophyllcontent and the ratio of chlorophyll to total nitrogen werehigher in summer than in other seasons. The results indicatethat nitrogen was used in a manner that better utilizes thevery weak light in summer and the higher light intensities inother seasons. The major component of the free amino acid poolwas asparagine, in every organ throughout the season, exceptfor the senescent leaves. Since asparagine has a high N:C ratio,we suppose that the asparagine-dominated amino acid pool isadvantageous in the carbon-limited environment of the forestfloor.Copyright 1994, 1999 Academic Press Free amino acid composition, total nitrogen, total soluble protein, photosynthesis, evergreen hemicryptophyte