Recognition of phospholipids in Streptomyces phospholipase D

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

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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 Gly 188 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.

Original languageEnglish
Pages (from-to)26143-26151
Number of pages9
JournalJournal of Biological Chemistry
Volume280
Issue number28
DOIs
Publication statusPublished - Jul 15 2005

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Phospholipase D
Streptomyces
Phospholipids
DNA Shuffling
Amino Acids
Amino Acid Motifs
Nucleophiles
Substrates
Gene Library
Spectrum Analysis
Spectroscopy
Kinetics
DNA
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Recognition of phospholipids in Streptomyces phospholipase D. / Uesugi, Yoshiko; Mori, Koichi; Arima, Jiro; Iwabuchi, Masaki; Hatanaka, Tadashi.

In: Journal of Biological Chemistry, Vol. 280, No. 28, 15.07.2005, p. 26143-26151.

Research output: Contribution to journalArticle

Uesugi, Y, Mori, K, Arima, J, Iwabuchi, M & Hatanaka, T 2005, 'Recognition of phospholipids in Streptomyces phospholipase D', Journal of Biological Chemistry, vol. 280, no. 28, pp. 26143-26151. https://doi.org/10.1074/jbc.M414319200
Uesugi, Yoshiko ; Mori, Koichi ; Arima, Jiro ; Iwabuchi, Masaki ; Hatanaka, Tadashi. / Recognition of phospholipids in Streptomyces phospholipase D. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 28. pp. 26143-26151.
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