Matthews Megan L, Krest Courtney M, Barr Eric W, Vaillancourt Frédéric H, Walsh Christopher T, Green Michael T, Krebs Carsten, Bollinger J Martin
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Biochemistry. 2009 May 26;48(20):4331-43. doi: 10.1021/bi900109z.
Aliphatic halogenases activate O(2), cleave alpha-ketoglutarate (alphaKG) to CO(2) and succinate, and form haloferryl [X-Fe(IV)O; X = Cl or Br] complexes that cleave aliphatic C-H bonds to install halogens during the biosynthesis of natural products by non-ribosomal peptide synthetases (NRPSs). For the related alphaKG-dependent dioxygenases, it has been shown that reaction of the Fe(II) cofactor with O(2) to form the C-H bond-cleaving ferryl complex is "triggered" by binding of the target substrate. In this study, we have tested for and defined structural determinants of substrate triggering (ST) in the halogenase, SyrB2, from the syringomycin E biosynthetic NRPS of Pseudomonas syringae B301D. As for other halogenases, the substrate of SyrB2 is complex, consisting of l-Thr tethered via a thioester linkage to a covalently bound phosphopantetheine (PPant) cofactor of a carrier protein, SyrB1. Without an appended amino acid, SyrB1 does not trigger formation of the chloroferryl intermediate state in SyrB2, even in the presence of free l-Thr or its analogues, but SyrB1 charged either by l-Thr (l-Thr-S-SyrB1) or by any of several non-native amino acids does trigger the reaction by as much as 8000-fold (for the native substrate). Triggering efficacy is sensitive to the structures of both the amino acid and the carrier protein, being diminished by 5-24-fold when the native l-Thr is replaced with another amino acid and by approximately 40-fold when SyrB1 is replaced with the heterologous carrier protein, CytC2. The directing effect of the carrier protein and consequent tolerance for profound modifications to the target amino acid allow the chloroferryl state to be formed in the presence of substrates that perturb the ratio of its two putative coordination isomers, lack the target C-H bond (l-Ala-S-SyrB1), or contain a C-H bond of enhanced strength (l-cyclopropylglycyl-S-SyrB1). For the latter two cases, the SyrB2 chloroferryl state so formed exhibits unprecedented stability (t(1/2) = 30-110 min at 0 degree C), can be trapped at high concentration and purity by manual freezing without a cryosolvent, and represents an ideal target for structural characterization. As initial steps toward this goal, extended X-ray absorption fine structure (EXAFS) spectroscopy has been used to determine the Fe-O and Fe-Cl distances and density functional theory (DFT) calculations have been used to confirm that the measured distances are consistent with the anticipated structure of the intermediate.
脂肪族卤化酶激活氧气，将α-酮戊二酸（αKG）裂解为二氧化碳和琥珀酸，并形成卤代高铁酰基[X-Fe(IV)O；X = Cl或Br]配合物，该配合物在非核糖体肽合成酶（NRPSs）参与的天然产物生物合成过程中裂解脂肪族碳氢键以引入卤素。对于相关的依赖αKG的双加氧酶，已表明亚铁辅因子与氧气反应形成裂解碳氢键的高铁酰基配合物是由靶底物的结合“触发”的。在本研究中，我们测试并确定了来自丁香假单胞菌B301D的丁香霉素E生物合成NRPS中的卤化酶SyrB2的底物触发（ST）的结构决定因素。与其他卤化酶一样，SyrB2的底物很复杂，由通过硫酯键连接到载体蛋白SyrB1的共价结合的磷酸泛酰巯基乙胺（PPant）辅因子上的L-苏氨酸组成。没有附加氨基酸时，即使存在游离的L-苏氨酸或其类似物，SyrB1也不会触发SyrB2中氯高铁酰基中间态的形成，但由L-苏氨酸（L-Thr-S-SyrB1）或几种非天然氨基酸中的任何一种充电的SyrB1确实能触发该反应，反应效率提高多达8000倍（对于天然底物）。触发效率对氨基酸和载体蛋白的结构都很敏感，当天然的L-苏氨酸被另一种氨基酸取代时，触发效率降低5至24倍，当SyrB1被异源载体蛋白CytC2取代时，触发效率降低约40倍。载体蛋白的导向作用以及对靶氨基酸进行深度修饰的耐受性使得在存在扰乱其两种假定配位异构体比例、缺乏靶碳氢键（L-Ala-S-SyrB1）或含有增强强度碳氢键（L-环丙基甘氨酰-S-SyrB1）的底物时仍能形成氯高铁酰基状态。对于后两种情况，如此形成的SyrB2氯高铁酰基状态表现出前所未有的稳定性（0℃下t(1/2)=30-110分钟），可以通过手动冷冻在无冷冻溶剂的情况下以高浓度和高纯度捕获，并且是结构表征的理想靶标。作为朝着这一目标迈出的初步步骤，扩展X射线吸收精细结构（EXAFS）光谱已用于确定Fe-O和Fe-Cl距离，密度泛函理论（DFT）计算已用于确认测量的距离与中间体的预期结构一致。
