Identification of a Novel Saxitoxin Analogue, 12β-Deoxygonyautoxin 3, in the Cyanobacterium, Anabaena circinalis (TA04).

Minowa Minowa,Konoki Konoki,Cho Cho,Yotsu-Yamashita Yotsu-Yamashita,Oshima Oshima

doi:10.3390/toxins11090539

Abstract

Saxitoxin (STX) and its analogues, the potent voltage-gated sodium channel blockers, are biosynthesized by freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates in the extract of the cyanobacterium, Anabaena circinalis (TA04), that are primarily produced during the early and middle stages in the biosynthetic pathway to produce STX. These findings allowed us to propose a putative biosynthetic pathway responsible for STX production based on the structures of these intermediates. In the present study, we identified 12β-deoxygonyautoxin 3 (12β-deoxyGTX3), a novel STX analogue produced by A. circinalis (TA04), by comparing the retention time and MS/MS fragmentation pattern with those of synthetic standards using LC–MS. The presence of this compound in A. circinalis (TA04) is consistent with stereoselective enzymatic oxidations at C11 and C12, and 11-O-sulfation, during the late stage of STX biosynthesis, as proposed in previous studies.

Highlights

Saxitoxin (STX (1), Figure 1) and its analogues, the potent voltage-gated sodium channel blockers [1], are known as paralytic shellfish toxins (PST) [2,3]
Concerning the O-sulfonation and N-sulfonation enzymes, Sako et al [10] purified and characterized a sulfotransferase specific to N-21 of STX and gonyautoxin (GTX) 2/3, and Yoshida et al [11] characterized a sulfotransferase specific to O-22 of 11-hydroxysaxitoxin; both enzymes were detected in the toxic marine dinoflagellate, Gymnodium catenatum
Using LC–MS, we identified a novel PST analogue in A. circinalis (TA04) extract, and the structure was determined to be 12β-deoxygonyautoxin 3 (12β-deoxyGTX3) (3) by comparison with synthetic standards

Summary

Introduction

Saxitoxin (STX (1), Figure 1) and its analogues, the potent voltage-gated sodium channel blockers [1], are known as paralytic shellfish toxins (PST) [2,3]. Several species of freshwater cyanobacteria and marine dinoflagellates have been identified to produce PST [2,3,4,5]. The biosynthetic pathway responsible for the synthesis of STX was first proposed by Shimizu et al [6] by conducting feeding experiments using stable isotope-labeled acetic acid and amino acids as essential substrates with PST producing cyanobacteria and dinoflagellates. The majority of the core set of genes (sxtA-sxtI, sxtP-sxtR, sxtS, and sxtU) were commonly identified in PST producing cyanobacteria [4]. We previously identified biosynthetic intermediates of STX, namely, Int-A’, Toxins 2019, 11, 539; doi:10.3390/toxins11090539 www.mdpi.com/journal/toxins

Methods

Results

Conclusion