Abstract
Saxitoxin, the most potent voltage-gated sodium channel blocker, is one of the paralytic shellfish toxins (PSTs) produced by cyanobacteria and dinoflagellates. Recently, putative biosynthetic genes of PSTs were reported in these microorganisms. We previously synthesized genetically predicted biosynthetic intermediates, Int-A’ and Int-C’2, and also Cyclic-C’ which was not predicted based on gene, and identified them all in the toxin-producing cyanobacterium Anabaena circinalis (TA04) and the dinoflagellate Alexandrium tamarense (Axat-2). This study examined the incorporation of 15N-labeled intermediates into PSTs (C1 and C2) in A. circinalis (TA04). Conversions from Int-A’ to Int-C’2, from Int-C’2 to Cyclic-C’, and from Int-A’ and Int-C’2 to C1 and C2 were indicated using high resolution-LC/MS. However, Cyclic-C’ was not converted to C1 and C2 and was detected primarily in the extracellular medium. These results suggest that Int-A’ and Int-C’2 are genuine precursors of PSTs, but Int-C’2 converts partially to Cyclic-C’ which is a shunt product excreted to outside the cells. This paper provides the first direct demonstration of the biosynthetic route towards saxitoxin and a shunt pathway.
Highlights
In our previous study[33,34], we synthesized the genetically predicted biosynthetic intermediates proposed by Kellmann et al.[31], Int-A’ (3) and Int-C’2 (4), as well as the related compound, Cyclic-C’ (7) (Fig. 1), and identified them in the paralytic shellfish toxins (PSTs)-producing cyanobacterium Anabaena circinalis (TA04)[15] and the dinoflagellate Alexandrium tamarense (Axat-2)[35,36,37] using high resolution (HR)-LC/MS
These compounds were not detected in the non-PST-producing cyanobacterium Anabaena circinalis (NIES-1645)[38] and dinoflagellate Alexandrium tamarense (UAT-014-009)[35,36,37], supporting the suggestion that these compounds are implicated in the STX biosynthesis
[2,6-15N2]Int-A’ (3′), [2,7-15N2]Int-C’2 (4′) and [3,9-15N2]Cyclic-C’ (7′) were independently administered into the culture medium of A. circinalis (TA04) at 5 μ M, and cultivation was continued for 7 days
Summary
[2,6-15N2]Arginine (2′), [2,6-15N2]Int-A’ (3′), [2,7-15N2]Int-C’2 (4′) and [3,9-15N2]Cyclic-C’ (7′) were synthesized from [2,5-15N2]L-ornithine by following the synthetic route we reported previously (Fig. 3)[33,34]. When [15N2]Cyclic-C’ (7′ ) was administered to the medium repetitively, the labeled ratio of C2 (9) did not increase at all until day 31 (Fig. 5b,c), suggesting that Cyclic-C’ (7) was not converted to C2 (9). The incorporation ratio of 15N-labels from [15N2]Int-C’2 (4′) into [15N2]C2 (9′) was approximately 17% at day 31 (Fig. 5a,c), even if [15N2]Int-C’2 (4′) was administered repetitively The concentration of total Cyclic-C’ (7) was much higher than the concentration of total Int-C’2 (4) in all experimental periods These results supported the above assumption that excessively administered Int-C’2 (4) (at 5 μ M in this experiment) was converted to Cyclic-C’ (7) and released from the cells into the culture medium, while a small part of the administered Int-C’2 (4) was converted to PSTs (C1 (8) and C2 (9)) (Fig. 6). This finding will accelerate efforts to clarify the full process of PSTs biosynthesis
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