Karl V. Wood

Developmental Regulation of Methyl Benzoate Biosynthesis and Emission in Snapdragon Flowers

In snapdragon flowers, the volatile ester methyl benzoate is the most abundant scent compound. It is synthesized by and emitted from only the upper and lower lobes of petals, where pollinators (bumblebees) come in contact with the flower. Emission of methyl benzoate occurs in a rhythmic manner, with maximum emission during the day, which correlates with pollinator activity. A novel S-adenosyl-l-methionine:benzoic acid carboxyl methyl transferase (BAMT), the final enzyme in the biosynthesis of methyl benzoate, and its corresponding cDNA have been isolated and characterized. The complete amino acid sequence of the BAMT protein has only low levels of sequence similarity to other previously characterized proteins, including plant O-methyl transferases. During the life span of the flower, the levels of methyl benzoate emission, BAMT activity, BAMT gene expression, and the amounts of BAMT protein and benzoic acid are developmentally and differentially regulated. Linear regression analysis revealed that production of methyl benzoate is regulated by the amount of benzoic acid and the amount of BAMT protein, which in turn is regulated at the transcriptional level.

Production of Se-methylselenocysteine in transgenic plants expressing selenocysteine methyltransferase.

BackgroundIt has become increasingly evident that dietary Se plays a significant role in reducing the incidence of lung, colorectal and prostate cancer in humans. Different forms of Se vary in their chemopreventative efficacy, with Se-methylselenocysteine being one of the most potent. Interestingly, the Se accumulating plant Astragalus bisulcatus (Two-grooved poison vetch) contains up to 0.6% of its shoot dry weight as Se-methylselenocysteine. The ability of this Se accumulator to biosynthesize Se-methylselenocysteine provides a critical metabolic shunt that prevents selenocysteine and selenomethionine from entering the protein biosynthetic machinery. Such a metabolic shunt has been proposed to be vital for Se tolerance in A. bisulcatus. Utilization of this mechanism in other plants may provide a possible avenue for the genetic engineering of Se tolerance in plants ideally suited for the phytoremediation of Se contaminated land. Here, we describe the overexpression of a selenocysteine methyltransferase from A. bisulcatus to engineer Se-methylselenocysteine metabolism in the Se non-accumulator Arabidopsis thaliana (Thale cress).ResultsBy over producing the A. bisulcatus enzyme selenocysteine methyltransferase in A. thaliana, we have introduced a novel biosynthetic ability that allows the non-accumulator to accumulate Se-methylselenocysteine and γ-glutamylmethylselenocysteine in shoots. The biosynthesis of Se-methylselenocysteine in A. thaliana also confers significantly increased selenite tolerance and foliar Se accumulation.ConclusionThese results demonstrate the feasibility of developing transgenic plant-based production of Se-methylselenocysteine, as well as bioengineering selenite resistance in plants. Selenite resistance is the first step in engineering plants that are resistant to selenate, the predominant form of Se in the environment.

Plant Phenylacetaldehyde Synthase Is a Bifunctional Homotetrameric Enzyme That Catalyzes Phenylalanine Decarboxylation and Oxidation

We have isolated and characterized Petunia hybrida cv. Mitchell phenylacetaldehyde synthase (PAAS), which catalyzes the formation of phenylacetaldehyde, a constituent of floral scent. PAAS is a cytosolic homotetrameric enzyme that belongs to group II pyridoxal 5′-phosphate-dependent amino-acid decarboxylases and shares extensive amino acid identity (∼65%) with plant l-tyrosine/3,4-dihydroxy-l-phenylalanine and l-tryptophan decarboxylases. It displays a strict specificity for phenylalanine with an apparent Km of 1.2 mm. PAAS is a bifunctional enzyme that catalyzes the unprecedented efficient coupling of phenylalanine decarboxylation to oxidation, generating phenylacetaldehyde, CO2, ammonia, and hydrogen peroxide in stoichiometric amounts.

Structure Elucidation of Natural Products by Mass Spectrometry

For over three decades, mass spectrometry has played a central role for the structure elucidation of natural products because it can be used to determine the molecular weights and elemental compositions of substances with a wide range of chemical and physical properties. In addition, if the molecular ion is formed with sufficient excitation energy, it will undergo a series of unimolecular reactions to give fragment ions, the relative abundances of which are normally unique to the structure of the molecular ion. By understanding the general rules governing the fragmentation reactions, the types of functional groups present, as well as the manner in which they are joined to form a unique molecular structure may be deduced from the mass spectrum.

Muricatacin: A simple biologically active acetogenin derivative from the seeds of annona muricata (annonaceae)

A biologically active constituent, bearing similarities to the annonacin group of Annonaceous acetogenins, has been isolated from the seeds of Annona muricata. The absolute configuration about the tetrahydrofuran ring of annonacin, which was also isolated, is proposed.

Methionine sulfoxide reductase A protects dopaminergic cells from Parkinson’s disease-related insults

Parkinsons disease (PD) is a neurologic disorder characterized by dopaminergic cell death in the substantia nigra. PD pathogenesis involves mitochondrial dysfunction, proteasome impairment, and alpha-synuclein aggregation, insults that may be especially toxic to oxidatively stressed cells including dopaminergic neurons. The enzyme methionine sulfoxide reductase A (MsrA) plays a critical role in the antioxidant response by repairing methionine-oxidized proteins and by participating in cycles of methionine oxidation and reduction that have the net effect of consuming reactive oxygen species. Here, we show that MsrA suppresses dopaminergic cell death and protein aggregation induced by the complex I inhibitor rotenone or mutant alpha-synuclein, but not by the proteasome inhibitor MG132. By comparing the effects of MsrA and the small-molecule antioxidants N-acetylcysteine and vitamin E, we provide evidence that MsrA protects against PD-related stresses primarily via methionine sulfoxide repair rather than by scavenging reactive oxygen species. We also demonstrate that MsrA efficiently reduces oxidized methionine residues in recombinant alpha-synuclein. These findings suggest that enhancing MsrA function may be a reasonable therapeutic strategy in PD.

Relative gas-phase acidities from triple quadrupole mass spectrometers

Abstract Chemical ionization of mixtures of aliphatic acids yields proton-bound dicarboxylate anions, R 1 CO 2 − ⋯H + ⋯ − O 2 CR 2 , which are mass-selected and subjected to collision-induced dissociation (CID) at 5–30 eV. The branching ratio for formation of the principal fragments, R 1 CO 2 − and R 2 CO 2 − , is used to follow internal energy deposition in the proton-bound anion as a function of collision energy and target pressure. Increased energy deposition is observed with increases in pressure and in translational energy, although the branching ratio also shows effects attributed to severe discrimination against the lighter fragment at pressures exceeding the limit for single collisions. These discrimination effects are most pronounced at low collision energy and are not observed (for argon target gas) at the higher energies. The relative abundances of the carboxylate fragments correlate with their relative proton affinities. In this, and in the fact that quantitative assignments of relative proton affinities are possible, the triple quadrupole methodology gives results which parallel those obtained using keV beams in sector mass spectrometers. Detailed similarities between the low- and high-energy studies include the anomalous behavior of formic acid in both and quantitative agreement in measured gas-phase acidities for valeric and hexanoic acid.

Near-Isogenic Lines of Maize Differing for Glycinebetaine

A series of near-isogenic glycinebetaine-containing and -deficient F8 pairs of Zea mays L. (maize) lines were developed. The pairs of lines differ for alternative alleles of a single locus; the wild-type allele conferring glycinebetaine accumulation is designated Bet1 and the mutant (recessive) allele is designated bet1. The near-isogenic lines were used to investigate whether glycinebetaine deficiency affects the pool size of the glycinebetaine precursor, choline, using a new method for glycinebetaine and choline determination: stable isotope dilution plasma desorption mass spectrometry. Glycinebetaine deficiency in maize was associated with a significant expansion of the free choline pool, but the difference in choline pool size was not equal to the difference in glycinebetaine pool size, suggesting that choline must down-regulate its own synthesis. Consistent with this, glycinebetaine deficiency was also associated with the accumulation of the choline precursor, serine. A randomly amplified polymorphic DNA marker was identified that detects the bet1 allele. In 62 F8 families tested the 10-mer primer 5[prime]-GTCCTCGTAG produced a 1.2-kb polymerase chain reaction product only when DNA from Bet1/bet1 or bet1/bet1 lines was used as template. All 26 homozygous Bet1/Bet1 F8 families tested were null for this marker.

The absolute configuration of trilobacin and trilobin, a novel highly potent acetogenin from the stem bark of Asimina triloba (Annonaceae)

Abstract Trilobacin ( 1 ) is the only previously reported Annonaceous acetogenin with the erythro relative configuration between the adjacent bis-tetrahydrofuran (THF) rings. The relative configuration in the adjacent bis-THF moiety of 1 was corrected to threo/trans/erythro/cis/threo from C-15 to C-24, and the absolute configuration was determined as 4 R , 15 R , 16 R , 19 R , 20 S , 23 R , 24 R and 36 S by advanced Mosher ester methodology and the COSY and NOESY spectral analyses of its 4,24-diacetate ( 1a ). Further investigation of the EtOH extract of the stem bark of the paw paw tree, Asimina triloba , monitoring for bioactivity by brine shrimp lethality, led to a novel acetogenin, trilobin ( 2 ). Spectral and chemical methods identified 2 as a 4-deoxy-10-hydroxy isomer of 1 . The absolute configuration of 2 was determined as 10 R , 15 R , 16 R , 19 R , 20 S , 23 R , 24 R , and 36 S by analyses of its Mosher ester derivatives. 2 was selectively cytotoxic among three human solid tumor cell lines with over a billion times the potency of adriamycin.

Characterization of betaines using electrospray MS/MS

Betaines are an important class of naturally occurring compounds that function as compatible solutes or osmoprotectants. Because of the permanent positive charge on the quaternary ammonium moiety, mass spectrometric analysis has been approached by desorption methods, including fast atom bombardment and plasma desorption mass spectrometry. Here we show that electrospray ionization MS gives comparable results to plasma desorption MS for a range of authentic betaine standards and betaines purified from plant extracts by ion exchange chromatography. A distinct advantage of electrospray ionization MS over plasma desorption MS is the capability of obtaining product ion spectra via MS/MS of selected parent ions, and hence structural information to discriminate between ions of identical mass.