Purification of a glutathione S-transferase and a glutathione conjugate-specific dehydrogenase involved in isoprene metabolism in Rhodococcus sp. strain AD45

A glutathione S transferase (GST) with activity toward 1,2-eposy-2-methyl-3-butene (isoprene monoxide) and cis-1,2-dichloroepoxyethane was purified from the isoprene-utilizing bacterium Rhodococcus sp. strain AD45, The homodimeric enzyme (two subunits of 27 kDa each) catalyzed the glutathione (GSH)-dependent ring opening of various epoxides, At 5 mM GSH, the enzyme followed Michaelis-Menten kinetics for isoprene monoxide and cis 1,2-dichloroepoxyethane, with V-max values of 66 and 2.4 mu mol min(-1) mg of protein(-1) and K-m values of 0.3 and 0.1 mM for isoprene monoside and cis-1,2-dichloroepoxyethane, respectively, Activities increased linearly with the GSH concentration up to 25 mM. H-1 nuclear magnetic resonance spectroscopy showed that the product of GSH conjugation to isoprene monoxide was 1-hydroxy-2-glutathionyl-2-methyl-3-butene (HGMB), Thus, nucleophilic attack of GSH occurred on the tertiary carbon atom of the epoxide ring. HGMB was further converted by an NAD(+)-dependent dehydrogenase, and this enzyme was also purified from isoprene-grown cells. The homodimeric enzyme (two subunits of 25 kDa each) showed a high activity for HGMB, whereas simple primary and secondary alcohols were not oxidized. The enzyme catalyzed the sequential oxidation of the alcohol function to the corresponding aldehyde and carboxylic acid and followed Michaelis-Menten kinetics,vith respect to NAD(+) and HGMB. The results suggest that the initial steps in isoprene metabolism are a monooxygenase-catalyzed conversion to isoprene monoxide, a GST-catalyzed conjugation to HGMB, and a dehydrogenase-catalyzed two-step oxidation to 2-glutathionyl-2-methyl-3-butenoic acid.</p

Morphological and Molecular Characterization of Orchid Fruit Development

Efficient seed dispersal in flowering plants is enabled by the development of fruits, which can be either dehiscent or indehiscent. Dehiscent fruits open at maturity to shatter the seeds, while indehiscent fruits do not open and the seeds are dispersed in various ways. The diversity in fruit morphology and seed shattering mechanisms is enormous within the flowering plants. How these different fruit types develop and which molecular networks are driving fruit diversification is still largely unknown, despite progress in eudicot model species. The orchid family, known for its astonishing floral diversity, displays a huge variation in fruit dehiscence types, which have been poorly investigated. We undertook a combined approach to understand fruit morphology and dehiscence in different orchid species to get more insight into the molecular network that underlies orchid fruit development. We describe fruit development in detail for the epiphytic orchid species Erycina pusilla and compare it to two terrestrial orchid species: Cynorkis fastigiata and Epipactis helleborine. Our anatomical analysis provides further evidence for the split carpel model, which explains the presence of three fertile and three sterile valves in most orchid species. Interesting differences were observed in the lignification patterns of the dehiscence zones. While C. fastigiata and E. helleborine develop a lignified layer at the valve boundaries, E. pusilla fruits did not lignify at these boundaries, but formed a cuticle-like layer instead. We characterized orthologs of fruit-associated MADS-domain transcription factors and of the Arabidopsis dehiscence-related genes INDEHISCENT (IND)/HECATE 3 (HEC3), REPLUMLESS (RPL) and SPATULA (SPT)/ALCATRAZ (ALC) in E. pusilla, and found that the key players of the eudicot fruit regulatory network appear well-conserved in monocots. Protein-protein interaction studies revealed that MADS-domain complexes comprised of FRUITFULL (FUL), SEPALLATA (SEP) and AGAMOUS (AG) /SHATTERPROOF (SHP) orthologs can also be formed in E. pusilla, and that the expression of HEC3, RPL, and SPT can be associated with dehiscence zone development similar to Arabidopsis. Our expression analysis also indicates differences, however, which may underlie fruit divergence

Structural Analysis of a Stereochemical Modification of Flavin Adenine Dinucleotide in Alcohol Oxidase from Methylotrophic Yeasts

Alcohol oxidase (MOX), a major peroxisomal protein of methanol-utilizing yeasts, contains two different forms of flavin adenine dinucleotide, one of which is identical with natural FAD whereas the other (mFAD) is a stereochemical modification of the natural coenzyme. This modification occurs spontaneously with FAD (but not FADH) bound to alcohol oxidase. mFAD was degraded with diphosphatase to provide authentic AMP and mFMN. The latter was degraded further with phosphatase to m-riboflavin. Analysis by 1H and 13C NMR spectroscopy of mFAD revealed that the isoalloxazine and adenine rings were intact and not modified structurally. However, significant differences were observed in the proton spectra in the sugar chains attached to the isoalloxazine ring (ribitol in the case of FAD). Similar observations were made for mFMN and m-riboflavin. Most striking in COSY spectra is the virtual absence of coupling between protons 2' and 3' in the sugar chain attached to the isoalloxazine ring, whereas this coupling is strong in the natural materials. Moreover, the nature of the coupling of proton 2' to protons 1a' and 1b' of the sugar chain is different in modified material. All these observations are consistent with the hypothesis that in modified cofactor the absolute configuration of carbon 2' of the sugar chain attached to the isoalloxazine ring has changed from R to S. This indicates the presence of an arabityl sugar chain rather than the ribitol present in natural FAD. A possible mechanism for this conversion is suggested.

5-Hydroxyaloesaponarin II, a minor blue pigment in an actinorhodin-negative mutant of Streptomyces coelicolor A3(2)

Blue pigmentation in Streptomyces coelicolor A3(2) is attributed to synthesis of the polyketide actinorhodin and its lactone derivative γ-actinorhodin, Therefore, actinorhodin-negative mutants show pigmentation other than blue. When the B22 mutant of S.coelicolor A3(2) [defective in the actVI-ORF1 gene coding for a putative keto( = oxo)reductase] was examined for its secondary metabolite content, the presence of aloesaponarin II (3,8-dihydroxy-1-methyl-9,10-anthraquinone) as the major pigment was confirmed. However, a substantial fraction of a red/blue (acid/alkaline) pigment was detected after separation on HPLC. MS and NMR analysis revealed its structure as 3,5,8-trihydroxy-1-methyl-9,10-anthraquinone. To our knowledge, this anthraquinone has not previously been reported in biological material. A possible route for biosynthesis of this compound is discussed.

Purification of a Glutathione S-Transferase and a Glutathione Conjugate-Specific Dehydrogenase Involved in Isoprene Metabolism in Rhodococcus sp. Strain AD45

A glutathione S-transferase (GST) with activity toward 1,2-epoxy-2-methyl-3-butene (isoprene monoxide) and cis-1,2-dichloroepoxyethane was purified from the isoprene-utilizing bacterium Rhodococcus sp. strain AD45. The homodimeric enzyme (two subunits of 27 kDa each) catalyzed the glutathione (GSH)-dependent ring opening of various epoxides. At 5 mM GSH, the enzyme followed Michaelis-Menten kinetics for isoprene monoxide and cis-1,2-dichloroepoxyethane, with V(max) values of 66 and 2.4 μmol min(−1) mg of protein(−1) and K(m) values of 0.3 and 0.1 mM for isoprene monoxide and cis-1,2-dichloroepoxyethane, respectively. Activities increased linearly with the GSH concentration up to 25 mM. (1)H nuclear magnetic resonance spectroscopy showed that the product of GSH conjugation to isoprene monoxide was 1-hydroxy-2-glutathionyl-2-methyl-3-butene (HGMB). Thus, nucleophilic attack of GSH occurred on the tertiary carbon atom of the epoxide ring. HGMB was further converted by an NAD(+)-dependent dehydrogenase, and this enzyme was also purified from isoprene-grown cells. The homodimeric enzyme (two subunits of 25 kDa each) showed a high activity for HGMB, whereas simple primary and secondary alcohols were not oxidized. The enzyme catalyzed the sequential oxidation of the alcohol function to the corresponding aldehyde and carboxylic acid and followed Michaelis-Menten kinetics with respect to NAD(+) and HGMB. The results suggest that the initial steps in isoprene metabolism are a monooxygenase-catalyzed conversion to isoprene monoxide, a GST-catalyzed conjugation to HGMB, and a dehydrogenase-catalyzed two-step oxidation to 2-glutathionyl-2-methyl-3-butenoic acid

CCDC 123044: Experimental Crystal Structure Determination

Related Article: A.M.Schoevaars, W.Kruizinga, R.W.J.Zijlstra, N.Veldman, A.L.Spek, B.L.Feringa|1997|J.Org.Chem.|62|4943|doi:10.1021/jo962210t,An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Composition of the Essential Oil From Roots and Rhizomes of Valeriana phu L. Growing Wild in Turkey

The volatile constituents isolated from roots and rhizomes of Valeriana phu L. were investigated by GC and GUMS (EI) analysis. The roots and rhizomes yielded 0.64% (v/w) essential oil on a dry weight basis. From the oil 70 compounds Could he identified with a valerenal isomer (11.3%), valerianol (3.1%), patchouli alcohol (2.9%) and valeranone (2.2%) as the main components. One new component was isolated and identified as 1-hydroxy-1,11,11-trimethyldecahydrocyclopropane azulene-10-one for the first time in V. phu