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J. Biol. Chem., Vol. 279, Issue 43, 44613-44620, October 22, 2004

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Hydrolytic Polyketide Shortening by Ayg1p, a Novel Enzyme Involved in Fungal Melanin Biosynthesis^*

Isao Fujii, Yoshinori Yasuoka, Huei-Fung Tsai¶, Yun C. Chang¶, K. J. Kwon-Chung¶, and Yutaka Ebizuka

From the Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan and the ^¶Laboratory of Clinical Infectious Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892-1882

The pentaketide 1,3,6,8-tetrahydroxynaphthalene (T4HN) is a key precursor of 1,8-dihydroxynaphthalene-melanin, an important virulence factor in pathogenic fungi, where T4HN is believed to be the direct product of pentaketide synthases. We showed recently the involvement of a novel protein, Ayg1p, in the formation of T4HN from the heptaketide precursor YWA1 in Aspergillus fumigatus. To investigate the mechanism of its enzymatic function, Ayg1p was purified from an Aspergillus oryzae strain that overexpressed the ayg1 gene. The Ayg1p converted the naphthopyrone YWA1 to T4HN with a release of the acetoacetic acid. Although Ayg1p does not show significant homology with known enzymes, a serine protease-type hydrolytic motif is present in its sequence, and serine-specific inhibitors strongly inhibited the activity. To identify its catalytic residues, site-directed Ayg1p mutants were expressed in Escherichia coli, and their enzyme activities were examined. The single substitution mutations S257A, D352A, and H380A resulted in a complete loss of enzyme activity in Ayg1p. These results indicated that the catalytic triad Asp³⁵²-His³⁸⁰-Ser²⁵⁷ constituted the active-site of Ayg1p. From a Dixon plot analysis, 2-acetyl-1,3,6,8-tetrahydroxynaphthalene was found to be a strong mixed-type inhibitor, suggesting the involvement of an acyl-enzyme intermediate. These studies support the mechanism in which the Ser²⁵⁷ at the active site functions as a nucleophile to attack the YWA1 side-chain 1'-carbonyl and cleave the carbon-carbon bond between the naphthalene ring and the side chain. Acetoacetic acid is subsequently released from the Ser²⁵⁷-O-acetoacetylated Ayg1p by hydrolysis. An enzyme with activity similar to Ayg1p in melanin biosynthesis has not been reported in any other organism.

Received for publication, June 17, 2004 , and in revised form, August 13, 2004.

^* This work was supported in part by Grant-in-aid for Scientific Research on Priority Area (A) 12045213 (to Y. E.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, Grant-in-aid for Scientific Research (C) 13836002 (to I. F.) from the Japan Society for the Promotion of Science (JSPS), and Grant-in-aid for Scientific Research (S) 15101007 (to Y. E.) from the JSPS. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to Dr. Heinz G. Floss on the occasion of his 70th birthday.

To whom correspondence should be addressed. Tel.: 81-3-5841-4743; Fax: 81-3-5841-4744; E-mail: ifujii{at}mol.f.u-tokyo.ac.jp.

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