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[交流] [优秀文章推荐] 水杨酸羟基化的代谢路径和调控机理领域取得新进展已有1人参与

Simplified Schematic of the Pathways for SA Modification and Catabolism in Arabidopsis

该研究通过体外酶活测定的方法筛选获得一个能催化水杨酸（SA）生成2,5-二羟基苯甲酸（2,5-DHBA）的酶S5H/DMR6，通过系统的酶动力学分析、植物代谢分析、表达分析和表型分析，揭示了S5H/DMR6 的生化特征及其维持SA动态平衡的分子机理，为深入研究SA羟基化代谢的生物学意义打下基础。

2,5-二羟基苯甲酸（2,5-DHBA），又名龙胆酸，是广泛存在于绿色植物的酚类化合物（Ibrahim, 1959，Nature），但是其合成途径尚不清楚。在拟南芥中存在2,3-二羟基苯甲酸（2,3-DHBA) 和2,5-二羟基苯甲酸(2,5-DHBA) 两种二羟基化苯甲酸，含量较高，被认为是SA的分解代谢的重要产物。SA 是一种重要的植物激素，在植物抗病、抗逆和生长发育中起到重要作用。负责催化SA生成2,3-DHBA的 S3H已被鉴定，该蛋白是一个2-酮戊二酸和亚铁依赖型加氧酶家族成员，其基因表达受SA和衰老信号诱导，起到延缓叶片衰老的作用（Zhang et al. 2013, PNAS)。鉴于2,3-DHBA 和2,5-DHBA的化学结构的相似性，推测2,5-DHBA也可能由其他2-酮戊二酸和亚铁依赖型加氧酶家族成员催化生成。该团队通过体外酶活检测的方法筛选一系列2-酮戊二酸和亚铁依赖型加氧酶，鉴定出能在体外催化SA生成2,5-DHBA的S5H酶。有意思的是该基因突变体是曾被鉴定为一个抗白粉病的突变体dmr6 (Van Damme et al., 2008, Plant J)。酶的动力学参数测定表明：与S3H相比较，S5H/DMR6 的Km值更低（大约低10倍），催化效率Kcat/Km更高（高约8倍），而且S5H/DMR6具有底物抑制活性，推测其在体内可能自我调控酶的活性而避免过度转化底物。

与此同时，该团队利用qRT-PCR 和GUS 报告基因检测了S5H/DMR6的表达特征，与S3H相比，S5H/DMR6对SA和病原菌诱导的响应更为灵敏并且在拟南芥整个生长发育阶段均表达，而S3H仅在衰老阶段，SA和病原菌诱导诱导条件下表达。

S5H/DMR6的T-DNA 插入突变体s5h表现出SA累积，植物稍微变小，对病原菌Pst. DC3000表现出强抗病和叶片早衰的表型，而s5hs3h双突变体表现出SA大量累积，植株显著矮小，超强抗病和严重叶片早衰的表型。该团队的系统研究结果表明水杨酸羟基化酶包括S5H/DMR6和S3H在拟南芥生长发育和抗病过程中协调作用，受SA诱导表达，通过对SA的羟基化使SA功能丧失，从而反馈调节SA的过度累积，维持SA动态平衡，影响植物抗病性、衰老等生命过程。

The phytohormone salicylic acid (SA) plays essential roles in biotic and abiotic responses, plant development, and leaf senescence. 2,5-Dihydroxybenzoic acid (2,5-DHBA or gentisic acid) is one of the most commonly occurring aromatic acids in green plants and is assumed to be generated from SA, but the enzymes involved in its production remain obscure. DMR6 (Downy Mildew Resistant 6, At5g24530) has been proven essential in plant immunity of Arabidopsis, but its biochemical properties are not well understood. Here, we report the discovery and functional characterization of DMR6 as a SA 5-hydroxylase (S5H) that catalyzes the formation of 2,5-DHBA by hydroxylating SA at the C5 position of its phenyl ring in Arabidopsis. S5H/DMR6 specifically converts SA to 2,5-DHBA in vitro and displays higher catalytic efficiency (Kcat/Km=4.96×104 M-1s-1) than the previously reported SA 3-hydroxylase (S3H, Kcat/Km=6.09×103 M-1s-1) for SA. Interestingly, S5H/DMR6 displays a substrate inhibition property that may enable automatic control of its enzyme activities. The s5h mutant and s5hs3h double mutant over accumulate SA and display phenotypes such as a smaller growth size, early senescence and a loss of susceptibility to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). S5H/DMR6 is sensitively induced by SA/pathogen treatment and is widely expressed from young seedlings to senescing plants, whereas S3H is more specifically expressed at the mature and senescing stages. Collectively, our results disclose the identity of the enzyme required for 2,5-DHBA formation and reveal a mechanism by which plants fine-tune SA homeostasis by mediating SA 5-hydroxylation.

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