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J. Biol. Chem., Vol. 280, Issue 28, 26143-26151, July 15, 2005

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Recognition of Phospholipids in Streptomyces Phospholipase D^*

Yoshiko Uesugi, Koichi Mori, Jiro Arima, Masaki Iwabuchi, and Tadashi Hatanaka

From the Research Institute for Biological Sciences, Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan

To investigate the contribution of amino acid residues to the enzyme reaction of Streptomyces phospholipase D (PLD), we constructed a chimeric gene library between two highly homologous plds, which indicated different activity in transphosphatidylation, using RIBS (repeat-length independent and broad spectrum) in vivo DNA shuffling. By comparing the activities of chimeras, six candidate residues related to transphosphatidylation activity were shown. Based on the above result, we constructed several mutants to identify the key residues involved in the recognition of phospholipids. By kinetic analysis, we identified that Gly¹⁸⁸ and Asp¹⁹¹ of PLD from Streptomyces septatus TH-2, which are not present in the highly conserved catalytic HXKXXXXD (HKD) motifs, are key amino acid residues related to the transphosphatidylation activity. To investigate the role of two residues in the recognition of phospholipids, the effects of these residues on binding to substrates were analyzed by surface plasmon spectroscopy. The result suggests that Gly¹⁸⁸ and Asp¹⁹¹ are involved in the recognition of phospholipids in correlation with the N-terminal HKD motif. Furthermore, this study also provides experimental evidence that the N-terminal HKD motif contains the catalytic nucleophile, which attacks the phosphatidyl group of the substrate.

Received for publication, December 20, 2004 , and in revised form, April 8, 2005.

^* 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.

Present address: Dept. of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, 3-1-1 Tsushima-naka, Okayama 700–8530, Japan.

To whom correspondence should be addressed: Research Institute for Biological Sciences, Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan. Tel.: 81-866-56-9452; Fax: 81-866-56-9454; E-mail: hatanaka{at}bio-ribs.com.

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