Visualizing the Interface of Biotin and Fatty Acid Biosynthesis through SuFEx Probes
Visualizing the Interface of Biotin and Fatty Acid Biosynthesis through SuFEx Probes
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DOI：
10.1021/jacs.3c10181
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发表时间：
2024-01-04
影响因子：
15
通讯作者：
Burkart，Michael D.
中科院分区：
文献类型：
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作者：
Chen，Aochiu;Re，Rebecca N.;Burkart，Michael D.
位点特异性共价结合为识别和理解蛋白质-蛋白质相互作用提供了有力的工具。在这项研究中，我们发现硫氟交换（SuFEx）弹头有效地交联大肠杆菌酰基载体蛋白（AcpP）与其合作伙伴BioF，一个关键的吡哆醛5′-磷酸（PLP）依赖性酶在生物素生物合成的早期步骤，通过靶向酪氨酸残基接近活性位点。我们通过MS/MS分析来自两个伴侣的肽来鉴定交联位点。我们通过蛋白质晶体学和突变研究进一步评估BioF-AcpP界面。在与AcpP相互作用的BioF表面残基中，三个关键的精氨酸残基似乎参与AcpP识别，使得庚二酰-AcpP可以作为PLP介导的催化的酰基供体。这些发现验证了BioF的进化获得功能，允许生物体通过选择性地与酸性AcpP残基形成盐桥的表面修饰直接从脂肪酸生物合成中构建生物素。
Site-specific covalent conjugation offers a powerful tool to identify and understand protein–protein interactions. In this study, we discover that sulfur fluoride exchange (SuFEx) warheads effectively crosslink theEscherichia coliacyl carrier protein (AcpP) with its partner BioF, a key pyridoxal 5′-phosphate (PLP)-dependent enzyme in the early steps of biotin biosynthesis by targeting a tyrosine residue proximal to the active site. We identify the site of crosslink by MS/MS analysis of the peptide originating from both partners. We further evaluate the BioF–AcpP interface through protein crystallography and mutational studies. Among the AcpP-interacting BioF surface residues, three critical arginine residues appear to be involved in AcpP recognition so that pimeloyl-AcpP can serve as the acyl donor for PLP-mediated catalysis. These findings validate an evolutionary gain-of-function for BioF, allowing the organism to build biotin directly from fatty acid biosynthesis through surface modifications selective for salt bridge formation with acidic AcpP residues.