In the aflatoxin biosynthetic pathway, 5-oxoaverantin (OAVN) cyclase, the cytosolic enzyme, catalyzes the reaction from OAVN to (2S,5S)-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.Aflatoxins are toxic, carcinogenic, and mutagenic secondary metabolites mainly produced by certain strains of Aspergillus flavus and Aspergillus parasiticus. Contamination by aflatoxins in food and feed is a serious problem in many areas of the world. The biosynthetic pathway of aflatoxin has been studied extensively, and most of the steps have been clarified (reviewed in references 12, 16, 18, 19, and 29). Many enzymes and the genes encoding the enzymes have been isolated, and most of the enzyme genes were found to comprise a huge cluster over 70 kb long in the fungal genome (16,25,27,29).We recently reported that two enzymes are involved in the pathway from 5Ј-hydroxyaverantin (HAVN) to averufin (AVR); HAVN dehydrogenase catalyzes the conversion of HAVN to 5Ј-oxoaverantin (OAVN), and OAVN cyclase catalyzes the next reaction from OAVN to AVR (13). These enzymes have been purified and characterized. The identity of HAVN dehydrogenase with the gene product of adhA (3) was confirmed by tryptic digestion and matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis (13). The molecular masses of natural OAVN cyclase and denatured OAVN cyclase are 158 kDa and 79 kDa, respectively.In this study, we determined the amino acid sequence of the enzyme to find the gene encoding the OAVN cyclase. Surprisingly, the N-terminal sequence of OAVN cyclase was the same as a stretch of the versiconal (VHOH) cyclase sequence that was deduced from the reported vbs gene (15). VHOH cyclase has been purified independently by Lin and Anderson (10) and by McGuire et al. (11). VHOH cyclase catalyzes the co...