The reaction of yeast cystathionine beta-synthase is rate-limited by the conversion of aminoacrylate to cystathionine

Our studies of the reaction mechanism of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are facilitated by the spectroscopic properties of the pyridoxal phosphate coenzyme that forms a series of intermediates in the reaction of L-serine and L-homocysteine to form L-cystathionine. To characterize these reaction intermediates, we have carried out rapid-scanning stopped-flow and single-wavelength stopped-flow kinetic measurements under pre-steady-state conditions, as well as circular dichroism and fluorescence spectroscopy under steady-state conditions. We find that the gem-diamine and external aldimine of aminoacrylate are the primary intermediates in the forward half-reaction with L-serine and that the external aldimine of aminoacrylate or its complex with L-homocysteine is the primary intermediate in the reverse half-reaction with L-cystathionine. The second forward half-reaction of aminoacrylate with L-homocysteine is rapid. No primary kinetic isotope effect was obtained in the forward half-reaction with L-serine. The results provide evidence (1) that the formation of the external aldimine of L-serine is faster than the formation of the aminoacrylate intermediate, (2) that aminoacrylate is formed by the concerted removal of the alpha-proton and the hydroxyl group of L-serine, and (3) that the rate of the overall reaction is rate-limited by the conversion of aminoacrylate to L-cystathionine. We compare our results with cystathionine beta-synthase with those of related investigations of tryptophan synthase and O-acetylserine sulfhydrylase.     

Abnormal germling development by brown rust and powdery mildew on cer barley mutants

The barley leaf rust fungus forms appressoria over host leaf stomata and penetrates via the stomatal pore. High levels of avoidance to leaf rust fungi have been described in some wild accessions of Hordeum species where a prominent wax layer on the stomata inhibits triggering of fungal appressorium differentiation. Leaf rust avoidance has not yet been found in H. vulgare. Since cuticular leaf waxes are implicated in the avoidance trait, we screened 27 eceriferum (cer) mutant lines of H. vulgare for avoidance to barley leaf rust. These mutations affect leaf waxes. Reduction in numbers of germ tubes forming appressoria over stomata was found in some lines, but the greatest reduction (ca 30%) was less than previously found in wild barley spp. or in an accession of H. chilense used here as a check. In one line (cer-zh654), avoidance was due to a combination of factors. Firstly, fewer germ tubes oriented towards stomata and so failed to contact them. Secondly, some germ tubes that encountered stomata did not form appressoria but over-grew them. In this line, therefore, the fungus tended to fail both to locate and to respond to stomata. The appressoria of barley powdery mildew form on leaf epidermal cells that they penetrate directly. On certain cer lines, a proportion of germlings of the barley powdery mildew fungus developed abnormally, suggesting that germlings failed to recognise and/or respond to the leaf surface waxes on these mutants.     

Characterization of Lr46, a gene conferring partial resistance to wheat leaf rust

Components of resistance conferred by the Lr46 gene, reported as causing "slow rusting" resistance to leaf rust in wheat, were studied and compared with the effects of Lr34 and genes for quantitative resistance in cv. Akabozu. Lr34 is a gene that confers non-hypersensitive type of resistance. The effect of Lr46 resembles that of Lr34 and other wheats reported with partial resistance. At macroscopic level, Lr46 produced a longer latency period than observed on the susceptible recurrent parent Lalbahadur, and a reduction of the infection frequency not associated with hypersensitivity. Microscopically, Lr46 increased the percentage of early aborted infection units not associated with host cell necrosis and decreased the colony size. The effect of Lr46 is comparable to that of Lr34 in adult plant stage, but in seedling stage its effect is weaker than that of Lr34.     

The phenotypic expression of QTLs for partial resistance to barley leaf rust during plant development

Partial resistance is generally considered to be a durable form of resistance. In barley, Rphq2, Rphq3 and Rphq4 have been identified as consistent quantitative trait loci (QTLs) for partial resistance to the barley leaf rust pathogen Puccinia hordei. These QTLs have been incorporated separately into the susceptible L94 and the partially resistant Vada barley genetic backgrounds to obtain two sets of near isogenic lines (NILs). Previous studies have shown that these QTLs are not effective at conferring disease resistance in all stages of plant development. In the present study, the two sets of QTL–NILs and the two recurrent parents, L94 and Vada, were evaluated for resistance to P. hordei isolate 1.2.1 simultaneously under greenhouse conditions from the first leaf to the flag leaf stage. Effect of the QTLs on resistance was measured by development rate of the pathogen, expressed as latency period (LP). The data show that Rphq2 prolongs LP at the seedling stage (the first and second leaf stages) but has almost no effect on disease resistance in adult plants. Rphq4 showed no effect on LP until the third leaf stage, whereas Rphq3 is consistently effective at prolonging LP from the first leaf to the flag leaf. The changes in the effectiveness of Rphq2 and Rphq4 happen at the barley tillering stage (the third to fourth leaf stages). These results indicate that multiple disease evaluations of a single plant by repeated inoculations of the fourth leaf to the flag leaf should be conducted to precisely estimate the effect of Rphq4. The present study confirms and describes in detail the plant development-dependent effectiveness of partial resistance genes and, consequently, will enable a more precise evaluation of partial resistance regulation during barley development.     

Biochemical and Spectroscopic Characterization of the Human Mitochondrial Amidoxime Reducing Components hmARC-1 and hmARC-2 Suggests the Existence of a New Molybdenum Enzyme Family in Eukaryotes

The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that is presumed to form the catalytical part of a three-component enzyme system, consisting of mARC, heme/cytochrome b₅, and NADH/FAD-dependent cytochrome b₅ reductase. mARC proteins share a significant degree of homology to the molybdenum cofactor-binding domain of eukaryotic molybdenum cofactor sulfurase proteins, the latter catalyzing the post-translational activation of aldehyde oxidase and xanthine oxidoreductase. The human genome harbors two mARC genes, referred to as hmARC-1/MOSC-1 and hmARC-2/MOSC-2, which are organized in a tandem arrangement on chromosome 1. Recombinant expression of hmARC-1 and hmARC-2 proteins in Escherichia coli reveals that both proteins are monomeric in their active forms, which is in contrast to all other eukaryotic molybdenum enzymes that act as homo- or heterodimers. Both hmARC-1 and hmARC-2 catalyze the N-reduction of a variety of N-hydroxylated substrates such as N-hydroxy-cytosine, albeit with different specificities. Reconstitution of active molybdenum cofactor onto recombinant hmARC-1 and hmARC-2 proteins in the absence of sulfur indicates that mARC proteins do not belong to the xanthine oxidase family of molybdenum enzymes. Moreover, they also appear to be different from the sulfite oxidase family, because no cysteine residue could be identified as a putative ligand of the molybdenum atom. This suggests that the hmARC proteins and sulfurase represent members of a new family of molybdenum enzymes.     

Molecular mechanism of 14-3-3 protein-mediated inhibition of plant nitrate reductase

14-3-3 proteins regulate key processes in eukaryotic cells including nitrogen assimilation in plants by tuning the activity of nitrate reductase (NR), the first and rate-limiting enzyme in this pathway. The homodimeric NR harbors three cofactors, each of which is bound to separate domains, thus forming an electron transfer chain. 14-3-3 proteins inhibit NR by binding to a conserved phosphorylation site localized in the linker between the heme and molybdenum cofactor-containing domains. Here, we have investigated the molecular mechanism of 14-3-3-mediated NR inhibition using a fragment of the enzyme lacking the third domain, allowing us to analyze electron transfer from the heme cofactor via the molybdenum center to nitrate. The kinetic behavior of the inhibited Mo-heme fragment indicates that the principal point at which 14-3-3 acts is the electron transfer from the heme to the molybdenum cofactor. We demonstrate that this is not due to a perturbation of the reduction potentials of either the heme or the molybdenum center and conclude that 14-3-3 most likely inhibits nitrate reductase by inducing a conformational change that significantly increases the distance between the two redox-active sites.     

Mapping resistance to powdery mildew in barley reveals a large-effect nonhost resistance QTL

Key message

Resistance factors against non-adapted powdery mildews were mapped in barley. Some QTLs seem effective only to non-adapted mildews, while others also play a role in defense against the adapted form.The durability and effectiveness of nonhost resistance suggests promising practical applications for crop breeding, relying upon elucidation of key aspects of this type of resistance. We investigated which genetic factors determine the nonhost status of barley (Hordeum vulgare L.) to powdery mildews (Blumeria graminis). We set out to verify whether genes involved in nonhost resistance have a wide effectiveness spectrum, and whether nonhost resistance genes confer resistance to the barley adapted powdery mildew. Two barley lines, SusBgt_SC and SusBgt_DC, with some susceptibility to the wheat powdery mildew B. graminis f.sp. tritici (Bgt) were crossed with cv Vada to generate two mapping populations. Each population was assessed for level of infection against four B. graminis ff.spp, and QTL mapping analyses were performed. Our results demonstrate polygenic inheritance for nonhost resistance, with some QTLs effective only to non-adapted mildews, while others play a role against adapted and non-adapted forms. Histology analyses of nonhost interaction show that most penetration attempts are stopped in association with papillae, and also suggest independent layers of defence at haustorium establishment and conidiophore formation. Nonhost resistance of barley to powdery mildew relies mostly on non-hypersensitive mechanisms. A large-effect nonhost resistance QTL mapped to a 1.4 cM interval is suitable for map-based cloning.