Yu C L, Brooks S, Li Y, Subramanian M, Summers R, Pope M
Proteomics Facility, University of Iowa, Iowa City, IA, United States.
University of Alabama, Tuscaloosa, AL, United States.
Methods Enzymol. 2017;586:379-411. doi: 10.1016/bs.mie.2016.11.003. Epub 2017 Jan 17.
Understanding the pathophysiology of genes and enzymes involved in caffeine metabolism can have extracurricular benefits, such as providing distinct methylxanthines as intermediates for pharmaceutical synthesis, and also improve environmental waste remediation. The strains Pseudomonas putida CBB5 and CES may provide insights into these applications because they may both be induced to degrade caffeine, yet the latter thrives in concentrations >8.0gL; threefold higher than any other bacteria. We took a novel approach toward identifying the enzymatic pathways in both Pseudomonas sp. CES and a deletion mutation of strain CBB5, which largely circumvented the need for exhaustive isolation of enzymes and the stepwise reconstitution of their activities to determine caffeine response elements. Here, we describe two optimized, rapid alternative strategies based on multiplexed SIL assays and demonstrate their application by discovering caffeine-degrading enzymes in the CES strain based on quantitative comparison between enriched lysate fractions drawn from bacterial proteomes grown in the absence and presence of caffeine. Comparisons were made using stable isotope dimethyl labeling and expression differences were substantiated by reciprocal labeling experiments. The role of the identified proteins in caffeine degradation was independently verified by genetic sequencing. Multiple new components of N-demethylase system were discovered within a fraction of the lysate enriched specifically for this activity. We also describe how to expand the biological context (and reduce systemic bias) by adapting the protocol for total lysate analysis. We combined off-line prefractionation with the speed and resolution advantages of the Orbitrap LUMOS. The global protocol revealed 2406 proteins 1789 of which were quantified between treatments revealing, among other insights, a new antagonistic degradation pathway for vanillin that is completely suppressed by caffeine treatment.
了解参与咖啡因代谢的基因和酶的病理生理学具有额外的益处，例如提供独特的甲基黄嘌呤作为药物合成的中间体，还能改善环境废物修复。恶臭假单胞菌CBB5和CES菌株可能为这些应用提供见解，因为它们都可能被诱导降解咖啡因，但后者在浓度>8.0gL的环境中生长旺盛；比其他任何细菌高出三倍。我们采用了一种新颖的方法来鉴定假单胞菌属CES菌株和CBB5菌株的缺失突变体中的酶促途径，这在很大程度上避免了对酶进行详尽分离以及逐步重建其活性以确定咖啡因反应元件的需求。在这里，我们描述了基于多重SIL分析的两种优化的快速替代策略，并通过基于从在有无咖啡因条件下生长的细菌蛋白质组中提取的富集裂解物组分之间的定量比较，在CES菌株中发现咖啡因降解酶来证明它们的应用。使用稳定同位素二甲基标记进行比较，并通过相互标记实验证实表达差异。通过基因测序独立验证了所鉴定蛋白质在咖啡因降解中的作用。在专门针对该活性富集的裂解物组分中发现了N-脱甲基酶系统的多个新组分。我们还描述了如何通过调整总裂解物分析方案来扩展生物学背景（并减少系统偏差）。我们将离线预分级分离与Orbitrap LUMOS的速度和分辨率优势相结合。全局方案揭示了2406种蛋白质，其中1789种在处理之间进行了定量，除其他见解外，还揭示了一种新的香草醛拮抗降解途径，该途径被咖啡因处理完全抑制。
