Insights into polyamine metabolism: homospermidine is double-oxidized in two discrete steps by a single copper-containing amine oxidase in pyrrolizidine alkaloid biosynthesis

Author:

Zakaria Mahmoud M12ORCID,Stegemann Thomas1ORCID,Sievert Christian1ORCID,Kruse Lars H1ORCID,Kaltenegger Elisabeth1ORCID,Girreser Ulrich3ORCID,Çiçek Serhat S4ORCID,Nimtz Manfred5ORCID,Ober Dietrich1ORCID

Affiliation:

1. Botanical Institute and Botanic Gardens, Kiel University, Kiel, Germany

2. Department of Pharmacognosy, Faculty of Pharmacy, Zagazig University, Zagazig 44519, Egypt

3. Department of Pharmaceutical and Medicinal Chemistry, Kiel University, Kiel, Germany

4. Department of Pharmaceutical Biology, Kiel University, Kiel, Germany

5. Cellular Proteome Research, Helmholtz Centre for Infection Research, Braunschweig, Germany

Abstract

Abstract Polyamines are important metabolites in plant development and abiotic and biotic stress responses. Copper-containing amine oxidases (CuAOs) are involved in the regulation of polyamine levels in the cell. CuAOs oxidize primary amines to their respective aldehydes and hydrogen peroxide. In plants, aldehydes are intermediates in various biosynthetic pathways of alkaloids. CuAOs are thought to oxidize polyamines at only one of the primary amino groups, a process frequently resulting in monocyclic structures. These oxidases have been postulated to be involved in pyrrolizidine alkaloid (PA) biosynthesis. Here, we describe the identification and characterization of homospermidine oxidase (HSO), a CuAO of Heliotropium indicum (Indian heliotrope), involved in PA biosynthesis. Virus-induced gene silencing of HSO in H. indicum leads to significantly reduced PA levels. By in vitro enzyme assays after transient in planta expression, we show that this enzyme prefers Hspd over other amines. Nuclear magnetic resonance spectroscopy and mass spectrometry analyses of the reaction products demonstrate that HSO oxidizes both primary amino groups of homospermidine (Hspd) to form a bicyclic structure, 1-formylpyrrolizidine. Using tracer feeding, we have further revealed that 1-formylpyrrolizidine is an intermediate in the biosynthesis of PAs. Our study therefore establishes that HSO, a canonical CuAO, catalyzes the second step of PA biosynthesis and provides evidence for an undescribed and unusual mechanism involving two discrete steps of oxidation that might also be involved in the biosynthesis of complex structures in other alkaloidal pathways.

Funder

German Egyptian Research Long-term fellowship

German Research Foundation to D.O. (Deutsche Forschungsgemeinschaft, DFG

Publisher

Oxford University Press (OUP)

Subject

Cell Biology,Plant Science

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