Post translational modifications of Trifolitoxin: a blue fluorescent peptide antibiotic.

Trifolitoxin (TFX, C41H63N15O15S) is a selective, ribosomally-synthesized, post-translationally modified, peptide antibiotic, produced by Rhizobium leguminosarum bv. trifolii T24. TFX specifically inhibits α-proteobacteria, including the plant symbiont Rhizobium spp., the plant pathogen Agrobacterium spp. and the animal pathogen Brucella abortus. TFX-producing strains prevent legume root nodulation by TFX-sensitive rhizobia. TFX has been isolated as a pair of geometric isomers, TFX1 and TFX2, which are derived from the biologically inactive primary amino acid sequence: Asp-Ile-Gly-Gly-Ser-Arg-Gln-Gly-Cys-Val-Ala. Gly-Cys is present as a thiazoline ring and the Arg-Gln-Gly sequence is extensively modified to a UV absorbing, blue fluorescent chromophore. The chromophore consists of a conjugated, 5-membered heterocyclic ring and side chain of modified glutamine.

Agrocinopine C, a Ti-plasmid-coded enzyme-product, is a 2-O, 6-O linked phosphodiester of D-Glucose and sucrose.

Agrocinopine C is a small molecule found in crown gall tumours induced by pathogenic Agrobacterium radiobacter carrying the tumour-inducing plasmid pTi Bo542. This phosphodiester opine was isolated (at 0.02 g/100 g fresh wt.) from sunflower (Helianthus annuus L.) galls. It is structurally related to agrocinopine A and is a glucose-2-phosphodiester linked to the C6-hydroxy-methyl group of the glucose moiety of sucrose. Sugar-2-phosphates are uncommon in plant tissues, whether transformed by Agrobacterium or not. 1H and 31P NMR signal multiplicity indicates five-fold anomeric complexity of agrocinopine C in solution, implying that the permeases taking up these sucrose-phosphodiesters could recognise any one of the five anomers. Data suggests that the open chain aldehyde forms of the 2-phosphorylated opines agrocinopine C and agrocinopine A and the corresponding phosphorylated glucose-2-phosphoramidate component of the antibiotic agrocin 84 play a central role in agrocin's selective toxicity to certain strains of Agrobacterium after uptake via Ti plasmid-encoded permeases.

Toward an understanding of mechanisms involved in non-polyphenol oxidase (Non-PPO) darkening in yellow alkaline noodles (YAN).

Asian noodles prepared from bread wheat flour darken over time due to a combination of polyphenol oxidase (PPO) activity and non-PPO effects. Although the enzymatic mechanism associated with the PPO reaction is well established, the non-PPO component consists of both physical (e.g., changes in surface properties) and chemical reactions. Variations in pH and solvents were used to gain a quantitative estimate of the contribution of physical and chemical components to non-PPO darkening in yellow alkaline noodles (YAN). In a set of five common high-PPO Australian wheat cultivars it was estimated that on average non-PPO darkening accounted for 69% of total darkening, with approximately two-thirds of this due to physical darkening and one-third had a chemical origin. Data from the chemical portion of non-PPO darkening is consistent with the presence of a PPO-like enzyme that oxidizes tyrosine, has a pH maximum of 8.1, and is inhibited by 50% methanol or ethanol but in the noodle is insensitive to PPO inhibitors such as tropolone. Therefore, with low-PPO and PPO-free wheat varieties becoming available, it may be possible to further reduce darkening in YAN by breeding for wheat varieties with low or zero levels of this PPO-like enzyme.

Impact of protein on darkening in yellow alkaline noodles.

Darkening in yellow alkaline noodles (YAN) was examined over a 24 h period in noodles made from 4 wheat varieties, including varieties with different levels of polyphenol oxidase (PPO) activity, selected to cover a range of protein levels. Noodles were made in the presence and absence of the PPO inhibitor, tropolone. The darkening was divided into two time periods: 0-4 h and 4-24 h. The first four hours was described by a composite rate equation, and this period was subdivided into two stages. The rate of darkening in the first stage was independent of both protein concentration and PPO activity. The amount of darkening (c), however, was highly dependent on protein concentration during this stage (-tropolone, r = 0.902; +tropolone, r = 0.905), but independent of PPO activity. The first stage darkening was a zero order reaction where additional protein does not increase the reaction rate, but when the protein supply has been depleted, the reaction stops. The rate of darkening during the first stage (k'(1) = 5.6 +/- 1.0) was similar to the rate of change in the protein structure (k'(1) = 6.5 +/- 1.3) as measured using the amide II band by infrared spectroscopy. This suggested that the first stage of darkening represents changes in light reflectance and absorbance caused by changes in hydrogen bonding rather than changes in covalent bonding. During the second stage of darkening, both the rate (k'(2)) and amount of darkening (DeltaL*(4h-c)) were significantly correlated with protein concentration (-tropolone, r = 0.465; +tropolone, r = 0.813), and in the absence of tropolone the amount of darkening was increased by PPO activity. The amount of darkening (DeltaL*(24h-4h)) during the second time period (4-24 h) (or third stage) was significantly correlated in the presence of tropolone (r = 0.375) and in the absence of tropolone (r = 0.428) with protein concentration. However, compared with earlier stages the response of non-PPO darkening during the third stage to change in protein concentration was smaller. Protein oxidation, or more specifically oxidation of tyrosine groups within the protein, appears to be the main mechanism involved in non-PPO darkening in YAN during the second and third stages with glutenin being the main reactant. Albumin and globulin are important substrates for PPO. No differences in darkening were detected in YAN made from the four varieties in the presence of tropolone; however, differences in YAN darkening were observed for the second and third stages due to site and year variation.

Physical-chemical analysis of non-polyphenol oxidase (non-PPO) darkening in yellow alkaline noodles.

Darkening in yellow alkaline noodles (YAN) was measured over 24 h in a high polyphenol oxidase (PPO) bread wheat ( Triticum aestivum L. cv. Tasman) and a very low PPO durum wheat ( Triticum durum cv. Kamilaroi). Over 24 h non-PPO darkening occurred across a range of pH 3.5-10.5, and in Tasman this was overlaid by darkening from PPO activity. The rate of darkening in YAN was separated into two main time periods, 0-4 and 4-24 h. The first 4 h of darkening was further divided into two stages using a composite first-order rate equation. Several specific inhibitors that partially inhibited non-PPO darkening were identified. These inhibitors, as well as the PPO inhibitors SHAM and tropolone, were used to analyze YAN darkening. The rate of the early stage of darkening was not altered by any inhibitors used; however, the magnitude of darkening was reduced by inhibitors specific for non-PPO darkening. Both the rate and extent of non-PPO darkening of the second stage of darkening were decreased in Tasman and Kamilaroi by inhibitors specific for non-PPO darkening, whereas both PPO inhibitors only decreased darkening in Tasman. The second and third stages of darkening showed similar characteristics. The third stage of darkening was examined in YAN made from Kamilaroi over a temperature range from -4 to 65 degrees C. It followed an Arrhenius relationship indicating non-PPO darkening during this stage was nonenzymatic. The inhibitor data suggested that the reactive component(s) was/were present in a reasonably high concentration(s) and that the soluble protein fraction was involved in the non-PPO darkening process.