W.Russell McLauchlan

Interactions defining the specificity between fungal xylanases and the xylanase-inhibiting protein XIP-I from wheat

We previously reported on the xylanase-inhibiting protein I (XIP-I) from wheat [McLauchlan, Garcia-Conesa, Williamson, Roza, Ravestein and Maat (1999), Biochem. J. 338, 441-446]. In the present study, we show that XIP-I inhibits family-10 and -11 fungal xylanases. The K(i) values for fungal xylanases ranged from 3.4 to 610 nM, but bacterial family-10 and -11 xylanases were not inhibited. Unlike many glycosidase inhibitors, XIP-I was not a slow-binding inhibitor of the Aspergillus niger xylanase. Isothermal titration calorimetry of the XIP-I-A. niger xylanase complex showed the formation of a stoichiometric (1:1) complex with a heat capacity change of -1.38 kJ x mol(-1) x K(-1), leading to a predicted buried surface area of approx. 2200+/-500 A(2) at the complex interface. For this complex with A. niger xylanase (K(i)=320 nM at pH 5.5), titration curves indicated that an observable interaction occurred at pH 4-7, and this was consistent with the pH profile of inhibition of activity. In contrast, the stronger complex between A. nidulans xylanase and XIP-I (K(i)=9 nM) led to an observable interaction across the entire pH range tested (3-9). Using surface plasmon resonance, we show that the differences in the binding affinity of XIP-I for A. niger and A. nidulans xylanase are due to a 200-fold lower dissociation rate k(off) for the latter, with only a small difference in association rate k(on).

Quercetin inhibits hydrogen peroxide-induced oxidation of the rat lens

Cataract results from oxidative damage to the lens. The mechanism involves disruption of the redox system, membrane damage, proteolysis, protein aggregation and a loss of lens transparency. Diet has a significant impact on cataract development, but the individual dietary components responsible for this effect are not known. We show that low micromolar concentrations of the naturally-occurring flavonoid, quercetin, inhibit cataractogenesis in a rat lens organ cultured model exposed to the endogenous oxidant hydrogen peroxide. Other phenolic antioxidants, (+)epicatechin and chlorogenic acid, are much less effective. Quercetin was active both when incubated in the culture medium together with hydrogen peroxide, and was also active when the lenses were pre-treated with quercetin prior to oxidative insult. Quercetin protected the lens from calcium and sodium influx, which are early events leading to lens opacity, and this implies that the non-selective cation channel is protected by this phenolic. It did not, however, protect against formation of oxidized glutathione resulting from H2O2 treatment. The results demonstrate that quercetin helps to maintain lens transparency after an oxidative insult. The lens organ culture/hydrogen peroxide (LOCH) model is also suitable for examining the effect of other dietary antioxidants.

Substrate (aglycone) specificity of human cytosolic β-glucosidase

Human cytosolic beta-glucosidase (hCBG) is a xenobiotic-metabolizing enzyme that hydrolyses certain flavonoid glucosides, with specificity depending on the aglycone moiety, the type of sugar and the linkage between them. Based upon the X-ray structure of Zea mays beta-glucosidase, we generated a three-dimensional model of hCBG by homology modelling. The enzyme exhibited the (beta/alpha)(8)-barrel fold characteristic of family 1 beta-glucosidases, with structural differences being confined mainly to loop regions. Based on the substrate specificity of the human enzymes, sequence alignment of family 1 enzymes and analysis of the hCBG structural model, we selected and mutated putative substrate (aglycone) binding site residues. Four single mutants (Val(168)-->Tyr, Phe(225)-->Ser, Tyr(308)-->Ala and Tyr(308)-->Phe) were expressed in Pichia pastoris, purified and characterized. All mutant proteins showed a decrease in activity towards a broad range of substrates. The Val(168)-->Tyr mutation did not affect K (m) on p -nitrophenyl ( p NP)-glycosides, but increased K (m) 5-fold on flavonoid glucosides, providing the first biochemical evidence supporting a role for this residue in aglycone-binding of the substrate, a finding consistent with our three-dimensional model. The Phe(225)-->Ser and Tyr(308)-->Ala mutations, and, to a lesser degree, the Tyr(308)-->Phe mutation, resulted in a drastic decrease in specific activities towards all substrates tested, indicating an important role of those residues in catalysis. Taken together with the three-dimensional model, these mutation studies identified the amino-acid residues in the aglycone-binding subsite of hCBG that are essential for flavonoid glucoside binding and catalysis.

The purification and immunocharacterisation of N-methylputrescine oxidase from transformed root cultures of Nicotiana tabacum L. cv SC58

The enzyme N-methylputrescine oxidase which catalyses the conversion of N-methylputrescine to N-methylpyrrolinium salt has been purified to homogeneity from transformed roots of Nicotiana tabacum L. cv SC58. The enzyme has an apparent sub-unit molecular weight of 53 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with gel-filtration studies, indicating that the native form is a dimer. The Km of the enzyme for N-methylputrescine has been estimated to be 0.1 mM. Polyclonal antibodies raised to the purified protein recognise one product in an immunoblot of a crude extract of transformed root tissue and will immunoprecipitate N-methylputrescine oxidase activity from such an extract. The antibodies also show a high degree of specificity in immunoblots of crude extracts of transformed root cultures from a range of other solanaceous and non-solanaceous species but do not cross-react with a partially purified preparation of pea-seedling diamine oxidase.

Diamine oxidation and alkaloid production in transformed root cultures of Nicotiana tabacum

Abstract Cadaverine, putrescine and N -methylputrescine were all oxidized by extracts of transformed roots of Nicotiana tabacum , the apparent K m values being, respectively, 2.75, 0.35 and 0.08 mM and the relative maximal oxidation rates, 100, 48 and 34%. For the same substrates, partially purified pea seedling diamine oxidase (DAO, EC 1.4.3.6) gave apparent K m values of 0.026, 0.040 and 0.046 mM, with relative maximal activities of 100, 65 and 65% respectively. For the Nicotiana extract, with equimolar substrate concentrations (1 and 0.1 mM), N -methylputrescine inhibited the oxidation of both cadaverine and putrescine, whereas the converse inhibitions did not occur. N -Methylputrescine (1 mM) caused only a modest stimulation of nicotine and nornicotine production when fed to transformed root cultures of N. tabacum , which was inhibited by equimolar cadaverine. Conversely, N -methylputrescine diminished the stimulation of anabasine production by cadaverine.