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Applied and Environmental Microbiology, June 2005, p. 2999-3006, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.2999-3006.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Aspergillus parasiticus Cyclase Catalyzes Two Dehydration Steps in Aflatoxin Biosynthesis

Emi Sakuno,^1,2, Ying Wen,^1,, Hidemi Hatabayashi,¹ Hatsue Arai,¹ Chiemi Aoki,¹ Kimiko Yabe,^1* and Hiromitsu Nakajima²

National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan,¹ Faculty of Agriculture, Tottori University, Koyama, Tottori 680-8553, Japan²

Received 6 October 2004/ Accepted 15 December 2004

In the aflatoxin biosynthetic pathway, 5'-oxoaverantin (OAVN) cyclase, the cytosolic enzyme, catalyzes the reaction from OAVN to (2'S,5'S)-averufin (AVR) (E. Sakuno, K. Yabe, and H. Nakajima, Appl. Environ. Microbiol. 69:6418-6426, 2003). Interestingly, the N-terminal 25-amino-acid sequence of OAVN cyclase completely matched an internal sequence of the versiconal (VHOH) cyclase that was deduced from its gene (vbs). The purified OAVN cyclase also catalyzed the reaction from VHOH to versicolorin B (VB). In a competition experiment using the cytosol fraction of Aspergillus parasiticus, a high concentration of VHOH inhibited the enzyme reaction from OAVN to AVR, and instead VB was newly formed. The recombinant Vbs protein, which was expressed in Pichia pastoris, showed OAVN cyclase activity, as well as VHOH cyclase activity. A mutant of A. parasiticus SYS-4 (= NRRL 2999) with vbs deleted accumulated large amounts of OAVN, 5'-hydroxyaverantin, averantin, AVR, and averufanin in the mycelium. These results indicated that the cyclase encoded by the vbs gene is also involved in the reaction from OAVN to AVR in aflatoxin biosynthesis. Small amounts of VHOH, VB, and aflatoxins also accumulated in the same mutant, and this accumulation may have been due to an unknown enzyme(s) not involved in aflatoxin biosynthesis. This is the first report of one enzyme catalyzing two different reactions in a pathway of secondary metabolism.

E.S. and Y.W. contributed equally to this work.

Present address: Department of Microbiology, College of Biological Science, China Agricultural University, Beijing 100094, China.