Deciphering enzymatic amine formation from glucosinolates in Brassica vegetables ‒ VEGAMIN

• Applicant: Dr. Franziska Hanschen
• Subject Area: Food Chemistry
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 524096542

Glucosinolates, which are found for example in cabbage, broccoli, radish or rocket, can be hydrolyzed enzymatically to form isothiocyanates which show cancer-preventive effects. These reactive and pungent-tasting compounds serve plants primarily as protection against pests and predators. In addition to isothiocyanates, however, nitriles or epithionitriles can also be released, depending on the enzymatic equipment of the plant and the glucosinolate structure. Due to their health promoting effects, it is desirable that such vegetables release mainly isothiocyanates. Recently, we discovered that isothiocyanates in cabbage (Brassica oleracea var. capitata) can be degraded to amines by an enzyme-like mechanism in raw vegetables. Amines can therefore represent major degradation products of glucosinolates in cabbage and may be more abundant than the corresponding isothiocyanate. None of the previously known microbial isothiocyanate hydrolases (ITCases) could be detected, nor was a plant protein identified that had similarity to described ITCases. So, a previously unknown plant enzyme appears to be responsible for amine formation. Therefore, the objectives of this project are I) to identify, isolate and characterize the isothiocyanate-hydrolyzing enzyme and II) to find out how widespread this degradation pathway is in Brassicaceae vegetables/plants and what role amine formation plays in different ontogenetic stages and tissues. To achieve these goals, first a rapid assay for isothiocyanate hydrolase activity will be developed. Then, plant proteins are fractionated and proteins from amine-forming fractions are identified by mass spectrometry. Candidate proteins will be are overexpressed in E. coli, the recombinant protein is isolated, and the ITCase is identified. The activity of the ITCase towards different substrates will then be investigated, and the influence of any cofactors on the activity will be evaluated. To better understand the importance of this amine formation pathway for the plant but also for nutrition, the distribution of ITCase-related amine formation in major Brassicaceae plants/vegetables will be investigated by ITCase activity screening and metabolite analysis, and ITCases will be characterized via proteomic analyses. In addition, the role of amine formation in different plant tissues and different ontogenetic stages will be investigated and correlated with ITCase expression. The results of this project significantly expand the understanding of enzymatic glucosinolate degradation and ultimately contribute to a better understanding of the nutritional importance of glucosinolates and their degradation products for humans but also their role for plants.

• DFG Programme: Research Grants
• Co-Investigators: Professor Philip Anthony Wigge, Ph.D.; Dr. Katja Witzel