Two enzymes involved in alkaloid biosynthesis production mechanisms in Aristolochia discovered

A research group led by Lecturer Yasuyuki Yamada and Professor Nobukazu Shitan from the Laboratory of Medicinal Cell Biology at Kobe Pharmaceutical University, Lecturer Kazuyoshi Terasaka from the Graduate School of Pharmaceutical Sciences at Nagoya City University, and Professor Fumihiko Sato (at the time of the research) from the Graduate School of Biostudies at Kyoto University announced that they have isolated two O-methyltransferases involved in the biosynthesis of "benzylisoquinoline alkaloids (BIAs)," natural organic compounds that include powerful bioactive substances such as aristolochic acids produced by plants of the genus Aristolochia, and have elucidated their functions. This is expected to lead to the elucidation of the alkaloid production mechanisms in these plants. The results were published in Plant Physiology and Biochemistry on December 13.

Plants of the genus Aristolochia produce various medicinal components and have been used as medicines since ancient times, but the aristolochic acids contained in them have been reported to have nephrotoxicity and carcinogenicity. In Japan, crude drugs containing aristolochic acids are not currently approved.

Aristolochic acids are classified as BIAs, represented by the analgesic morphine and the antibacterial intestinal drug berberine. BIAs include many rare and useful compounds with pharmacological activity.

On the other hand, due to the characteristic structure of BIAs in which a benzyl group is attached to an isoquinoline ring skeleton, the production mechanism within plants is not completely understood. There is an expectation that clarifying this will make it possible to search for new pharmacological activities through genetic modification.

Therefore, in this study, cultured cells and redifferentiated plants of A. debilis, which grows wild in various parts of Japan, were obtained. RNA sequencing analysis was performed to comprehensively search for gene groups expected to be involved in the biosynthesis of aristolochic acids and other BIAs. From these, two O-methyltransferase genes (OMT genes) that make a significant contribution to BIA skeletal formation were isolated.

These two O-methyltransferases (AdOMT1, AdOMT2) were expressed in E.coli, the purified proteins were reacted with various BIA substrates, and the products were analyzed.

As a result, it was found that both enzymes recognize a wide range of BIAs centered on the 1-benzylisoquinoline type, and AdOMT1 in particular may have the activity to methylate hydroxyl groups at multiple sites.

Furthermore, when kinetic analysis of the reactions of both enzymes was performed, it was revealed that AdOMT1 has particularly high overall catalytic function for norcoclaurine and norlaudanosoline, and AdOMT2 has high overall catalytic function for coclaurine.

The researchers also performed three-dimensional structure prediction using AlphaFold2 and docking prediction with substrates.

Yamada commented: "Aristolochic acids produced by plants of the genus Aristolochia have an extremely unique skeleton among benzylisoquinoline alkaloids, and there are still many remaining mysteries surrounding their biosynthetic mechanism. In the future, we expect that the isolation of enzyme genes that work downstream in the biosynthetic pathway will progress further, and the complete picture of aristolochic acid biosynthesis will be elucidated in detail at the molecular level."

Journal Information
Publication: Plant Physiology and Biochemistry
Title: Molecular characterization of two O-methyltransferases involved in benzylisoquinoline alkaloid biosynthesis in Aristolochia debilis
DOI: 10.1016/j.plaphy.2025.110956