J. Korean Soc. Appl. Biol. Chem. 54(4), 633-636 (2011) Short Communication A Peptide Produced by Pseudomonas tolaasi , Tolaasin Binds to Metal Ions Sunhee Lee 1 , Geunhyeong Jo 1 , Doseok Hwang 1 , Yoonkyung Woo 1 , Younggiu Lee 1 , Yeonjoong Yong 1 , Kyungrai Kang 1 , Jiye Hyun 1 , Young-Kee Kim 2 , Dong-Woon Kim 3 , and Yoongho Lim 1 * 1 Division of Bioscience and Biotechnology, BMIC, RCD, Konkuk University, Seoul 143-701, Reapublic of Korea 2 Department of Agricultural Chemistry, Chungbuk National University, Chungbuk 361-763, Republic of Korea 3 Swine science division, National Institute of Animal Science, RDA, Cheonan 330-801, Republic of Korea Received March 10, 2011; Accepted April 12, 2011 Brown blotch disease in mushrooms is caused by Pseudomonas tolaasin, which produces a peptide toxin, tolaasin I, and zinc ion inhibits the channel formed by tolaasin I. NMR experiments revealed that zinc, sodium, and calcium ions can bind to tolaasin I and their binding position on tolaasin I is the lactone ring. Key words: calcium ion, Pseudomonas tolaasi, sodium ion, tolaasin, zinc ion The brown blotch disease of mushrooms is found in Agaricus bisporus, Pleurotus ostreatus, and Flammulina velutipes. It is caused by Pseudomonas tolaasin, a species of Gram-negative soil bacteria producing peptide toxin, tolaasin [Cho et al., 2007]. Should the brown blotch disease occur during the fruit body formation, whole tissues of young mushrooms can be infected by tolaasin. In comparison, in the case of adult mushrooms, often brown spots can be observed in their tissues. Since this disease is highly contagious, people concentrate most of their efforts on its prevention. Of which, fumigation of cultivation house and sterilization of water are two widely used methods, due to consumers’ rejection of antibiotics. As mentioned above, considering tolaasin is the cause of brown blotch, the prevention of such can also be achieved by controlling tolaasin release. Tolaasin induces hemolysis [Cho et al., 2000] and seven analogues of tolaasin have been identified to date, which are I, II, A, B, C, D, and E [Bassarello et al., 2004]. Of these, tolaasin I is the main virulence factor, and it is a peptide consisting of 18 amino acids with 1985 Da [Nutkins et al., 1991]. Its N-terminal is acylated by β- hydroxyoctanoic acid and the C-terminal has a ring composed of D-alloThr 14 and lactone. Tolaasin forms pores within the cellular membrane, and therefore disrupts its structure [Brodey et al., 1991]. Tolaasin I contains 11 D-amino acids and its sequence is β- hydroxyoctanoyl- DBut 1 - DPro- DSer- DLeu- DVal- DSer- DLeu- DVal-L Val-DGln-LLeu-DVal-DBut-DalloThr-LIle-LHse- DDab-LLys 18 , where DBut, LHse, and DDab denote Z- dehydroaminobutyric acid, L-homoserine, and D-2,4- diaminobutyric acid, respectively [Coraiola et al., 2006] Tolaasin forms two types of ion channels and both ion channels are inhibited by zinc ions [Cho and Kim, 2003]. The presence of the ion channels is proven by the relationship of the slope conductance of the ion channel with a linear current vs. voltage. Therefore, authors assumed that zinc ions bind to tolaasin. In order to prove the hypothesis, nuclear magnetic resonance (NMR) spectroscopy was applied since a compound or an ion with inhibitory activity against tolaasin I must be able to bind to it, and any binding would normally lead to a chemical shift change in the 1 H-NMR spectrum. Tolaasin I was isolated from the fermentation broth of P. tolaasii according to the methods previously described by Shirata et al. [1995] and Cho et al. [2007]. Tolaasin I was dissolved in 150 μL of methanol-d 4 and its concentration was adjusted to be 5 mM. This solution was transferred into 2.5 mm NMR tube. Furthermore, 50 mM zinc chloride solution was prepared in methanol-d4. Following on this zinc chloride solution was added into the tolaasin I solution in the NMR tube from 50 μL to 250 μL and their 1 H-NMR data were collected at every 50 μL addition interval. The NMR experiments were carried out on a Bruker Avance 400 instrument (Bruker, Karlsruhe, Germany). The experimental details were as described in Lee et al. [Lee et al., 2009; Koh, 2010]. The stacked plot of the 1 H- *Corresponding author Phone: +82-2-450-3760; Fax: +82-2-454-3760 E-mail: yoongho@konkuk.ac.kr http://dx.doi.org/10.3839/jksabc.2011.096