Recognition of Phospholipids in Streptomyces Phospholipase D

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

doi:10.1074/jbc.m414319200

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 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.

Highlights

Phospholipase D (PLD,1 EC 3.1.4.4) is a ubiquitous enzyme found in bacteria, fungi, plants, and mammals [1]
We identified that Gly188 and Asp191 of phospholipase D (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
Construction of Chimeric PLDs—The chimeric pld genes were obtained when the plasmid pACTIS4b (Fig. 2A) for RIBS in vivo DNA shuffling was transformed into the ssb-3 mutant

Summary

Recognition of Phospholipids in Streptomyces Phospholipase D*

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 (repeatlength independent and broad spectrum) in vivo DNA shuffling. We found two amino acid residues related to the thermostability of Streptomyces PLD containing HKD motifs using RIBS (repeat-length independent and broad spectrum) in vivo DNA shuffling. This system is a novel method of random chimeragenesis based on the combination of highly frequent deletion formation in the Escherichia coli ssb-3 strain with an rpsL-based chimera selection system [22]. To investigate amino acid residues related to the PLD-catalyzed reaction, we constructed chimeras between TH-2PLD and PLDP from a distinct Streptomyces sp., which has been used extensively in transphosphatidylation reactions

EXPERIMENTAL PROCEDURES

RESULTS

Km kcat

TABLE II Kinetic parameters for interactions of chimeras C and

DISCUSSION