Biochemical investigation of the tryptophan biosynthetic enzyme anthranilate phosphoribosyltransferase in plants.

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-08-31 DOI:10.1016/j.jbc.2023.105197
Miriam Li, Hisham Tadfie, Cameron G Darnell, Cynthia K Holland
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Abstract

While mammals require the essential amino acid tryptophan (Trp) in their diet, plants and microorganisms synthesize Trp de novo. The five-step Trp pathway starts with the shikimate pathway product, chorismate. Chorismate is converted to the aromatic compound anthranilate, which is then conjugated to a phosphoribosyl sugar in the second step by anthranilate phosphoribosyltransferase (PAT1). As a single-copy gene in plants, all fixed carbon flux to indole and Trp for protein synthesis, specialized metabolism, and auxin hormone biosynthesis proceeds through PAT1. While bacterial PAT1s have been studied extensively, plant PAT1s have escaped biochemical characterization. Using a structure model, we identified putative active site residues that were variable across plants and kinetically characterized six PAT1s (Arabidopsis thaliana (thale cress), Citrus sinensis (sweet orange), Pistacia vera (pistachio), Juglans regia (English walnut), Selaginella moellendorffii (spike moss), and Physcomitrium patens (spreading earth-moss)). We probed the catalytic efficiency, substrate promiscuity, and regulation of these six enzymes and found that the C. sinensis PAT1 is highly specific for its cognate substrate, anthranilate. Investigations of site-directed mutants of the A. thaliana PAT1 uncovered an active site residue that contributes to promiscuity. While Trp inhibits bacterial PAT1 enzymes, the six plant PAT1s that we tested were not modulated by Trp. Instead, the P. patens PAT1 was inhibited by tyrosine, and the S. moellendorffii PAT1 was inhibited by phenylalanine. This structure-informed biochemical examination identified variations in activity, efficiency, specificity, and enzyme-level regulation across PAT1s from evolutionarily diverse plants.

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植物色氨酸生物合成酶邻氨基苯甲酸磷酸核糖转移酶的生化研究。
哺乳动物在饮食中需要必需的氨基酸色氨酸,而植物和微生物则从头合成色氨酸。五步Trp途径从莽草酸途径产物chorismate开始。Chorismate转化为芳香族化合物邻氨基苯甲酸,然后在第二步中通过邻氨基苯磷酸核糖转移酶(PAT1)与磷酸核糖结合。作为植物中的单拷贝基因,吲哚和Trp用于蛋白质合成、专门代谢和生长素生物合成的所有固定碳通量都通过PAT1进行。虽然细菌的PAT1已经被广泛研究,但植物的PAT1还没有得到生化表征。使用结构模型,我们鉴定了假定的活性位点残基,这些残基在不同植物中是可变的,并对六种PAT1(拟南芥(水芹)、柑橘(甜橙)、阿月浑子(开心果)、胡桃(英国胡桃)、卷柏(刺苔)和斑叶藻(铺展土苔))进行了动力学表征。我们探讨了这六种酶的催化效率、底物混杂性和调节,发现中华鳖PAT1对其同源底物邻氨基苯甲酸盐具有高度特异性。对拟南芥PAT1位点定向突变体的研究发现了一个导致滥交的活性位点残基。虽然Trp抑制细菌PAT1酶,但我们测试的六种植物PAT1没有受到Trp的调节。相反,P.patens PAT1受到酪氨酸的抑制,S.moellendorfii PAT1受到苯丙氨酸的抑制。这种基于结构的生物化学检查确定了进化多样性植物的PAT1在活性、效率、特异性和酶水平调节方面的变化。
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