Absolute configuration of aflastatin A, a specific inhibitor of aflatoxin production by Aspergillus parasiticus.

Aflastatin A (1) is a specific inhibitor of aflatoxin production by Aspergillus parasiticus. It has the novel structure of a tetramic acid derivative with a long alkyl side chain. The absolute configurations of 29 chiral centers contained in 1 were chemically elucidated in this study. First, four small fragment molecules were prepared from 1 or its methyl ether (2), and their absolute structures were assigned as N-methyl-D-alanine, (2S,4R)-2, 4-dimethyl-1,6-hexanediol dibenzoate, (R)-3-hydroxydodecanoic acid, and (R)-1,2,4-butanetriol tribenzoate. Next, an acyclic fragment molecule 3 with 13 chiral centers was obtained from 1 by NaIO(4) oxidation, and its relative stereochemistry was elucidated by J-based configuration analysis. By analyzing coupling constants of (3)J(H,H) and (2,3)J(C,H) and ROE data, the relative configuration of 3 was verified. Finally, by further J-based configuration analysis using a fragment molecule 7 prepared from 2 with 28 chiral carbons, all relative configurations in the alkyl side chain of 1 were clarified. By connecting these relative configurations with the absolute configurations of first four fragment molecules, the absolute stereochemistry of 1 was fully determined.

Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region.

The cellular target of leptomycin B (LMB), a nuclear export inhibitor, has been identified as CRM1 (exportin 1), an evolutionarily conserved receptor for the nuclear export signal of proteins. However, the mechanism by which LMB inhibits CRM1 still remains unclear. CRM1 in a Schizosaccharomyces pombe mutant showing extremely high resistance to LMB had a single amino acid replacement at Cys-529 with Ser. The mutant gene, named crm1-K1, conferred LMB resistance on wild-type S. pombe, and Crm1-K1 no longer bound biotinylated LMB. (1)H NMR analysis showed that LMB bound N-acetyl-L-cysteine methyl ester through a Michael-type addition, consistent with the idea that LMB binds covalently via its alpha, beta-unsaturated delta-lactone to the sulfhydryl group of Cys-529. When HeLa cells were cultured with biotinylated LMB, the only cellular protein bound covalently was CRM1. Inhibition by N-ethylmaleimide (NEM), an alkylating agent, of CRM1-mediated nuclear export probably was caused by covalent binding of the electrophilic structure in NEM to the sulfhydryl group of Cys-529, because the crm1-K1 mutant showed the normal rate for the export of Rev nuclear export signal-bearing proteins in the presence of not only LMB but also NEM. These results show that the single cysteine residue determines LMB sensitivity and is selectively alkylated by LMB, leading to CRM1 inactivation.

Involvement of afsA in A-factor biosynthesis as a key enzyme.

The afsA gene of Streptomyces griseus has been postulated to encode a key enzyme for A-factor biosynthesis from primary metabolites commonly present in Streptomyces strains. Escherichia coli cells harboring afsA under the control of the T7 promoter specified distinct A-factor activity in the culture broth, as determined by induction of streptomycin production and aerial mycelium and spore formation in an A-factor-deficient S. griseus mutant strain. Production of the substance(s) having A-factor activity was inhibited by cerulenin, an inhibitor of fatty acid biosynthesis. These observations suggest that afsA encodes a key enzyme in the A-factor biosynthetic pathway in which a beta-keto acid derived from fatty acid biosynthesis and a glycerol derivative serve as precursors.