Nigel C. Veitch

Substrate binding and catalysis in heme peroxidases

Peroxidase-catalysed reactions are being analysed at an increasingly advanced level of structural and mechanistic sophistication. A significant development in this respect has been the long-anticipated solution of crystal structures for several plant peroxidases and a fungal peroxidase complexed to benzhydroxamic acid. New insights into peroxide binding and catalysis have been obtained through site-directed mutagenesis, a technique also crucial to recent progress in understanding the diversity of substrate interaction sites associated with peroxidases from different sources.

Bacterial resistance modifying agents from Lycopus europaeus.

As part of an ongoing project to identify plant natural products which modulate bacterial multidrug resistance (MDR), bioassay-guided isolation of an extract of Lycopus europaeus yielded two new isopimarane diterpenes, namely methyl-1alpha-acetoxy-7alpha 14alpha-dihydroxy-8,15-isopimaradien-18-oate (1) and methyl-1alpha,14alpha-diacetoxy-7alpha-hydroxy-8,15-isopimaradien-18-oate (2). The structures were established by spectroscopic methods. These compounds and several known diterpenes were tested for in vitro antibacterial and resistance modifying activity against strains of Staphylococcus aureus possessing the Tet(K), Msr(A), and Nor(A) multidrug resistance efflux mechanisms. At 512 microg/ml none of the compounds displayed any antibacterial activity but individually in combination with tetracycline and erythromycin, a two-fold potentiation of the activities of these antibiotics was observed against two strains of S. aureus that were highly resistant to these agents due to the presence of the multidrug efflux mechanisms Tet(K) (tetracycline resistance) and Msr(A) (macrolide resistance).

External flavones in sweet basil, Ocimum basilicum, and related taxa

Abstract Sixteen accessions of Ocimum basilicum L. belonging to different cultivars and varieties, and one accession each of O. x citriodorum Vis. and O. minimum L., were examined for their external leaf flavonoids. The O. basilicum samples showed the presence of one or two major flavone aglycones, which were identified as salvigenin and nevadensin, and up to 10 minor ones: cirsileol, cirsilineol, eupatorin, apigenin, acacetin, genkwanin, apigenin 7,4′-dimethyl ether, cirsimaritin, ladenein and gardenin B. No trace was found of xanthomicrol, the only lipophilic flavone so far reported from O. basilicum . There were appreciable differences amongst the accessions in total flavonoid concentration and in their nevadensin / salvigenin ratios, which, on the whole, were not correlated with morphological characters. The surface flavonoid profile of O. x citriodorum was very similar to those of the O. basilicum plants studied, but that of O. minimum was significantly different. Only 4′-methyl ethers of apigenin derivatives were detected in the latter taxon, whereas apigenin and luteolin derivatives in which the 4′-hydroxyls are free were absent. Furthermore, the ratio of nevadensin / salvigenin was much higher in O. minimum than in O. basilicum or O. x citriodorum . These differences support the treatment of O. minimum as a separate species rather than as a variety of O. basilicum .

Structural determinants of plant peroxidase function

The classical plant peroxidases are a well-studied group of heme-containing enzymes for which many different functions have been proposed. In the majority of plant species investigated they occur as distinctive isoenzymes which can be constitutive or induced in response to external factors such as wounding, stress and attack by pathogens. More than 70 peroxidase isoenzymes are predicted to occur in Arabidopsis thaliana alone, according to recent analysis of the complete peroxidase gene family of this model plant. Understanding this enzymatic diversity and its functional significance is a major focus of structural and mechanistic studies of plant peroxidases. The three-dimensional structures of plant peroxidases from Arabidopsis, barley, horseradish, peanut and soybean have now been determined by X-ray crystallography together with the structures of several catalytic intermediates and substrate complexes that are relevant to enzyme function. On this basis, specific roles for particular amino acid residues and structural motifs or regions have been proposed or in some cases, confirmed. Some of these have been investigated experimentally using site-directed mutagenesis and other techniques. An overview of recent developments will be presented that reflects our current understanding of structure and function in this important group of enzymes.

Dihydroisocoumarins and a tetralone from Cytospora eucalypticola

Two dihydroisocoumarins, 3,5-dimethyl-8-hydroxy-7-methoxy-3,4-dihydroisocoumarin and 3,5-dimethyl-8-methoxy-3,4-dihydroisocoumarin were isolated from a culture filtrate of Cytospora eucalypticola, together with three known dihydroisocoumarins and a tetralone derivative. Their structures were determined by spectroscopic methods. These isocoumarins are mildly antifungal, and antibacterial towards gram positive bacteria. A known compound, 5-hydroxymethylmellein, showed mild antifeedant activity towards Spodoptera littoralis.

Leaf flavonoid glycosides as chemosystematic characters in Ocimum

Abstract Thirty-one accessions of nine species belonging to three subgenera of Ocimum (basil, family Lamiaceae) were surveyed for flavonoid glycosides. Substantial infraspecific differences in flavonoid profiles of the leaves were found only in O. americanum , where var. pilosum accumulated the flavone C -glycoside, vicenin-2, which only occurred in trace amounts in var. americanum and was not detected in cv. Sacred. The major flavonoids in var. americanum and cv. Sacred, and also in all other species investigated for subgenus Ocimum , were flavonol 3- O -glucosides and 3- O -rutinosides. Many species in subgenus Ocimum also produced the more unusual compound, quercetin 3- O -(6″- O -malonyl)glucoside, and small amounts of flavone O -glycosides. The level of flavonol glycosides produced was reduced significantly in glasshouse-grown plants, but levels of flavone glycosides were unaffected. A single species investigated from subgenus Nautochilus , O. lamiifolium , had a different flavonoid glycoside profile, although the major compound was also a flavonol O -glycoside. This was identified as quercetin 3- O -xylosyl(1‴→2″)galactoside, using NMR spectroscopy. The species investigated from subgenus Gymnocimum , O . tenuiflorum (= O. sanctum ), was characterised by the accumulation of flavone O -glycosides. These were isolated, and identified as the 7- O -glucuronides of luteolin and apigenin. Luteolin 5- O -glucoside was found in all nine species of Ocimum studied, and is considered to be a key character for the genus.

The chemotaxonomic significance of two bioactive caffeic acid esters, nepetoidins A and B, in the Lamiaceae.

A survey of leaf surface constituents in the family Lamiaceae using HPLC with diode array detection revealed the presence of two characteristic phenolic compounds in many species. The distribution of these phenolics in the Lamiaceae was found to be of taxonomic significance, as they were present in the great majority of species investigated for the subfamily Nepetoideae, including representatives of the well-known genera of culinary herbs, mint, rosemary, sage, thyme and basil. In contrast, they were absent from species of the other subfamilies of Lamiaceae studied and from the related families Verbenaceae, Scrophulariaceae, Acanthaceae and Buddlejaceae. The compounds were isolated from Plectranthus crassus and identified by NMR spectroscopy as the known caffeic acid esters (Z,E)-[2-(3,5-dihydroxyphenyl)ethenyl] 3-(3,4-dihydroxyphenyl)-2-propenoate and (Z,E)-[2-(3,4-dihydroxyphenyl)ethenyl] 3-(3,4-dihydroxyphenyl)-2-propenoate, for which the trivial names nepetoidins A and B are proposed. The presence of this pair of caffeic acid esters adds another character to the chemical, palynological and embryological features distinguishing the Nepetoideae from the other subfamilies of Lamiaceae and related families, and supports the view that the Nepetoideae are a specialised and monophyletic group within the family. Nepetoidin B was shown to have a greater antioxidant activity than gallic, rosmarinic and caffeic acids, and showed activity as an insect phagostimulant. Both compounds were antifungal.

Insect antifeedant furanocoumarins from Tetradium daniellii

The dried fruits of Tetradium daniellii yielded a new linear furanocoumarin, 5-(6-hydroxy-3,7-dimethylocta-2,7-dienyloxy)psoralen, together with six other structurally related furanocoumarins. A similar chemical profile was recorded by HPLC analysis of a fragment of T. daniellii fruit obtained from an historic herbarium voucher specimen collected in September 1917 during an expedition to Yunnan province, China. Four of the compounds identified caused a potent feeding deterrent effect towards larvae of Spodoptera littoralis and Heliothis virescens.

Bisdesmosidic saponins from Securidaca longepedunculata roots: evaluation of deterrency and toxicity to coleopteran storage pests.

Powdered dry root bark of Securidaca longepedunculata was mixed with maize and cowpea and effectively reduced the numbers of Sitophilus zeamais and Callosobruchus maculatus emerging from these commodities, respectively, more than 9 months after treatment. This effect was reciprocated in grain treated with a methanol extract of the root bark, indicating that compounds were present that were oviposition deterrents or directly toxic to the adults or larvae. Two new bisdesmosidic saponins, 3-O-beta-D-glucopyranosyl-28-O-(alpha-L-arabinopyranosyl-(1 --> 3)-beta-D-xylopyranosyl-(1 --> 4)[beta-D-apiofuranosyl-(1 --> 3)]-alpha-L-rhamnopyranosyl-(1 --> 2)-[4-O-(4-methoxycinnamoyl-beta-D-fucopyranosyl)])-medicagenic acid (securidacaside A) and 3-O-beta-D-glucopyranosyl-28-O-(alpha-L-arabinopyranosyl-(1 --> 3)-beta-D-xylopyranosyl-(1 --> 4)[beta-D-apiofuranosyl-(1 --> 3)]-alpha-L-rhamnopyranosyl-(1 --> 2)-[4-O-(3,4,5-trimethoxy-(E)-cinnamoyl-beta-D-fucopyranosyl)])-medicagenic acid (securidacaside B), were isolated from the methanol extract of the roots of S. longepedunculata and characterized by spectroscopic methods. Securidacaside A, which occurred as (E)- and (Z)-regioisomers, showed deterrency and toxicity toward C. maculatus and S. zeamais and could contribute to the biological activity of the methanol extract. The potential to optimize the use of this plant for stored product protection using water extracts, which would be appropriate technology for target farmers, is discussed.

Flavonoid cellobiosides from Salvia uliginosa

Abstract Two new flavone glycosides with an unusual interglycosidic linkage have been isolated from the petals of Salvia uliginosa and identified by NMR spectroscopy as apigenin 7-O- β- d -glucopyranosyl-(1→ → 4″)-β- d -glucopyranoside and apigenin 7-O- β- d -glucopyranosyl-(1→ → 4″)-β- d -glucopyranoside -4′-O- β- d -glucopyranoside . The NMR and UV spectra of these compounds were compared with those of the known compound, apigenin 7,4′-O,O- di-β- d -glucopyranoside , which was isolated from the same source.