Crystal structures of Yersinia enterocolitica salicylate synthase and its complex with the reaction products salicylate and pyruvate

The salicylate synthase, Irp9, from Yersinia enterocolitica is involved in the biosynthesis of the siderophore yersiniabactin. It is a bifunctional enzyme that forms salicylate and pyruvate from chorismate and water via the intermediate isochorismate. Here we report the first crystal structure of Irp9 and also of its complex with the reaction products salicylate and pyruvate at 1.85 A and 2.1 A resolution, respectively. Like other members of the chorismate-utilizing enzyme family, e.g. the TrpE subunit of anthranilate synthase and the PabB subunit of 4-amino-4-deoxychorismate synthase, Irp9 has a complex alpha/beta fold. The crystal structure of Irp9 contains one molecule each of phosphate and acetate derived from the crystallization buffer. The Irp9-products complex structure was obtained by soaking chorismate into Irp9, demonstrating that the enzyme is still catalytically active in the crystal. Both structures contain Mg(2+) in the active site. There is no evidence of the allosteric tryptophan binding site found in TrpE and PabB. Mutagenesis of Glu240, His321 and Tyr372 provided some insight into the mechanism of the two transformations catalyzed by Irp9. Knowledge of the structure of Irp9 will guide the search for potent inhibitors of salicylate formation, and hence of bacterial iron uptake, which is directly related to the virulence of Yersinia.     

Methyl salicylate production in tomato affects biotic interactions

The role of methyl salicylate (MeSA) production was studied in indirect and direct defence responses of tomato (Solanum lycopersicum) to the spider mite Tetranychus urticae and the root‐invading fungus Fusarium oxysporum f. sp. lycopersici, respectively. To this end, we silenced the tomato gene encoding salicylic acid methyl transferase (SAMT). Silencing of SAMT led to a major reduction in SAMT expression and MeSA emission upon herbivory by spider mites, without affecting the induced emission of other volatiles (terpenoids). The predatory mite Phytoseiulus persimilis, which preys on T. urticae, could not discriminate between infested and non‐infested SAMT‐silenced lines, as it could for wild‐type tomato plants. Moreover, when given the choice between infested SAMT‐silenced and infested wild‐type plants, they preferred the latter. These findings are supportive of a major role for MeSA in this indirect defence response of tomato. SAMT‐silenced tomato plants were less susceptible to a virulent strain of F. oxysporum f. sp. lycopersici, indicating that the direct defense responses in the roots are also affected in these plants. Our studies show that the conversion of SA to MeSA can affect both direct and indirect plant defence responses.     

不同诱导处理番茄植株对西花蓟马保护酶活性的影响

研究了西花蓟马取食茉莉酸、水杨酸甲酯、机械损伤、虫伤处理诱导的番茄植株对其虫体保护酶活性的影响。研究发现,不同处理的番茄植株对西花蓟马过氧化物酶(POD)、过氧化氢酶(CAT)和超氧化物歧化酶(SOD)3种保护酶的活性有明显的影响。只有西花蓟马取食茉莉酸处理的番茄植株24 h,POD活性明显升高,取食其它处理及在其它时间下,POD活性与对照没有明显的变化或活性受到抑制。取食水杨酸甲酯处理的番茄植株6 h和24 h,西花蓟马CAT活性均受到激发;取食虫害植株的3个时间段下,西花蓟马酶活性一直受到抑制;西花蓟马取食茉莉酸处理及机械损伤处理番茄植株,CAT酶活性在任何时间下都没有明显的变化或受到抑制。取食水杨酸诱导处理的番茄植株,西花蓟马SOD活性在6 h和24 h明显升高,36 h明显下降;但取食其它处理的SOD酶活性均在36 h明显升高。结果说明西花蓟马3种保护酶活性在取食不同处理诱导的番茄植株的不同时间下各不相同,说明保护酶活性的变化与不同诱导处理及时间密切相关。     

Expression and characterization of PhzE from P. aeruginosa PAO1: aminodeoxyisochorismate synthase involved in pyocyanin and phenazine-1-carboxylate production

PhzE from Pseudomonas aeruginosa catalyzes the first step in the biosynthesis of phenazine-1-carboxylic acid, pyocyanin, and other phenazines, which are virulence factors for Pseudomonas species. The reaction catalyzed converts chorismate into aminodeoxyisochorismate using ammonia supplied by a glutamine amidotransferase domain. It has structural and sequence homology to other chorismate-utilizing enzymes such as anthranilate synthase, isochorismate synthase, aminodeoxychorismate synthase, and salicylate synthase. Like these enzymes, it is Mg^2 + dependent and catalyzes a similar S_N2" nucleophilic substitution reaction. PhzE catalyzes the addition of ammonia to C2 of chorismate, as does anthranilate synthase, yet unlike anthranilate synthase it does not catalyze elimination of pyruvate from enzyme-bound aminodeoxyisochorismate. Herein, the cloning of the phzE gene, high level expression of active enzyme in E. coli, purification, and kinetic characterization of the enzyme is presented, including temperature and pH dependence. Steady-state kinetics give K_chorismate = 20 ± 4 μM, K_Mg^2 + = 294 ± 22 μM, K_L-gln = 11 ± 1 mM, and k_cat = 2.2 ± 0.2 s^− 1 for a random kinetic mechanism. PhzE can use NH₄⁺ as an alternative nucleophile, while Co^2 + and Mn^2 + are alternative divalent metals.     

Infection of Arabidopsis by cucumber mosaic virus triggers jasmonate-dependent resistance to aphids that relies partly on the pattern-triggered immunity factor BAK1

Many aphid-vectored viruses are transmitted nonpersistently via transient attachment of virus particles to aphid mouthparts and are most effectively acquired or transmitted during brief stylet punctures of epidermal cells. In Arabidopsis thaliana, the aphid-transmitted virus cucumber mosaic virus (CMV) induces feeding deterrence against the polyphagous aphid Myzus persicae. This form of resistance inhibits prolonged phloem feeding but promotes virus acquisition by aphids because it encourages probing of plant epidermal cells. When aphids are confined on CMV-infected plants, feeding deterrence reduces their growth and reproduction. We found that CMV-induced inhibition of growth as well as CMV-induced inhibition of reproduction of M. persicae are dependent upon jasmonate-mediated signalling. BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) is a co-receptor enabling detection of microbe-associated molecular patterns and induction of pattern-triggered immunity (PTI). In plants carrying the mutant bak1-5 allele, CMV induced inhibition of M. persicae reproduction but not inhibition of aphid growth. We conclude that in wildtype plants CMV induces two mechanisms that diminish performance of M. persicae: a jasmonate-dependent and PTI-dependent mechanism that inhibits aphid growth, and a jasmonate-dependent, PTI-independent mechanism that inhibits reproduction. The growth of two crucifer specialist aphids, Lipaphis erysimi and Brevicoryne brassicae, was not affected when confined on CMV-infected A. thaliana. However, B. brassicae reproduction was inhibited on CMV-infected plants. This suggests that in A. thaliana CMV-induced resistance to aphids, which is thought to incentivize virus vectoring, has greater effects on polyphagous than on crucifer specialist aphids.     

水杨酸钠对小鼠下丘核神经元GABAα、NR1表达及听反应的影响

目的:探讨水杨酸钠给药对小鼠下丘核神经元GABAα、NR1表达及听反应的影响。方法:实验将36只成年健康昆明小鼠分三组(n=12):A组:对照组;B组:水杨酸钠给药组(450mg.kg-1.d-1);C组:水杨酸钠(450mg.kg-1.d-1)+利多卡因(10mg.kg-1.d-1)组。各组均经腹腔注射给药,A组给以同等剂量的生理盐水,所有动物自由饮食。给药15 d后进行检测。采用RT-PCR技术检测小鼠下丘核区GABAα、NR1mRNA的表达;采用细胞外电生理记录技术研究下丘核区神经元在水杨酸钠给药后强度-发放率函数、强度-潜伏期函数和频率调谐曲线的变化。结果:①水杨酸钠组的下丘核区GABAαmRNA表达较对照组明显降低(P<0.05);水杨酸钠+利多卡因组与对照组比较无显著性差异(P>0.05);水杨酸钠组NR1mRNA表达较对照组明显升高(P<0.01),水杨酸钠+利多卡因组与对照组比较有显著性差异(P<0.05);②水杨酸钠给药后发放率和潜伏期函数呈非单调性改变,而神经元的调谐曲线从狭窄型向宽阔型转变;③水杨酸钠+利多卡因组发放率和潜伏期函数呈非单调性改变,而神经元的调谐曲线从狭窄型向宽阔型转变例数显著减少(P<0.05,P<0.01)。结论:①水杨酸钠引起实验小鼠下丘核GABAαmRNA表达下调,同时又使小鼠下丘核NR1mRNA表达增高;②水杨酸钠给药后发放率和潜伏期函数呈非单调性改变,而神经元的调谐曲线从狭窄型向宽阔型转变③利多卡因有逆转水杨酸钠给药后发放率和潜伏期函数呈非单调性改变和神经元的调谐曲线从狭窄型向宽阔型转变的作用。     

蓟马取食、机械损伤以及外源水杨酸甲酯和茉莉酸对菜豆叶片防御酶活性的影响

【目的】探讨菜豆对昆虫取食防御反应的生化机制。【方法】研究了西花蓟马Frankliniella occidentalis取食、机械损伤以及外源水杨酸甲酯（MeSA）和茉莉酸（JA）处理后菜豆叶片防御酶活性的变化。【结果】西花蓟马取食、机械损伤及MeSA和JA处理均能明显提高过氧化物酶（POD）的活性,前2种处理POD活性在72 h上升到最高峰,而后2种处理则在48 h达到最高峰。蛋白酶抑制剂（PI）活性在西花蓟马取食后升高最明显。JA途径关键酶脂氧合酶（LOX）和多酚氧化酶（PPO）的活性在西花蓟马取食、机械损伤和JA诱导处理均升高,但外源MeSA诱导处理则不能诱导它们的活性(P>0.05)。SA途径的关键酶苯丙氨酸解氨酶（PAL）在西花蓟马取食和机械损伤后均有一个先升高后下降的过程,外源MeSA诱导只在24 h引起PAL活性升高,其余时间下和对照没有明显的区别,外源JA诱导未能引起PAL活性的显著变化(P>0.05)。西花蓟马取食、JA和MeSA诱导以及机械损伤均能诱导β-1,3 葡聚糖酶（PR-2）活性上升(P<0.05)。【结论】结果说明,不同处理可诱导菜豆植株产生明显的防御反应,但酶活性的变化与处理方式和处理时间有关。