Tài liệu Báo cáo khoa học: Sensor of phospholipids inStreptomycesphospholipase D pdf

Sensor of phospholipids in Streptomyces phospholipase D

Yoshiko Uesugi, Jiro Arima, Masaki Iwabuchi and Tadashi Hatanaka

Research Institute for Biological Sciences (RIBS), Okayama, Japan

Phospholipase D (PLD; EC 3.1.4.4) catalyzes phos￾pholipid hydrolysis and phosphatidyl transfer

(Fig. 1A). This is a ubiquitous and important enzyme

involved in signal transduction in mammals [1,2].

Streptomyces PLDs can be categorized into two types:

one is an iron-containing enzyme, such as that from

Streptomyces chromofuscus (chromofuscus PLD) for

which tightly bound iron is necessary for its catalytic

activity [3]; and the other is a member of the PLD

superfamily whose hallmark is the possession of two

catalytic HxKxxxxD (HKD) motifs [4–6]. Because

enzymes of the latter type have a simple structure

containing two HKD motifs, they are useful as a

suitable model of mammalian PLDs.

A study of the chemical modification of PLD from

Streptomyces sp. PMF (PMFPLD) suggested that Lys,

not His, is essential for PLD activity [7]. Iwasaki et al.

[8] revealed that two HKD motifs are essential for the

activity, using the N- and C-terminal halves of Strep￾tomyces PLD. Furthermore, Leiros et al. [9] showed

that His170 in the N-terminal HKD motif of PMFPLD

acts as the initial nucleophile that attacks the phospho￾rus atom of the substrate, on the basis of the crystal

structures of PMFPLD. Previously, using two Strep￾tomyces PLDs in repeat-length independent and broad

spectrum (RIBS) in vivo DNA shuffling, we constructed

a random chimera library to investigate the recognition

of phospholipids by Streptomyces PLD. We revealed

that the N-terminal HKD motif contains the nucleo￾phile, using an inactive chimera and surface plasmon

resonance (SPR) analysis [10].

To date, the functions of the HKD motifs in cata￾lytic mechanisms have been extensively studied

[11–13]. At present, PLD-catalyzed reactions are con￾sidered to consist of two steps: first, the formation

of a covalently linked phosphatidyl enzyme interme￾diate via the His residue of the N-terminus HKD

motif; and second, the hydrolysis or transphosphati￾dylation of the intermediate by a water or alcohol

molecule (Fig. 1A).

As mentioned above, previous experimental studies

have focused on the relationship between HKD motifs

Keywords

phospholipase D; phospholipid; substrate

recognition; SPR; Streptomyces

Correspondence

T. Hatanaka, Research Institute for

Biological Sciences (RIBS), Okayama,

7549-1 Kibichuo-cho, Kaga-gun, Okayama

716-1241, Japan

Fax: +81 866 56 9454

Tel: +81 866 56 9452

E-mail: [email protected]

(Received 12 January 2007, revised 14

March 2007, accepted 22 March 2007)

doi:10.1111/j.1742-4658.2007.05802.x

Recently, we identified Ala426 and Lys438 of phospholipase D from Strep￾tomyces septatus TH-2 (TH-2PLD) as important residues for activity, sta￾bility and selectivity in transphosphatidylation. These residues are located

in a C-terminal flexible loop separate from two catalytic HxKxxxxD

motifs. To study the role of these residues in substrate recognition, we eval￾uated the affinities of inactive mutants, in which these residues were substi￾tuted with Phe and His, toward several phospholipids by SPR analysis. By

substituting Ala426 and Lys438 with Phe and His, respectively, the inactive

mutant showed a much stronger interaction with phosphatidylcholine and

a weaker interaction with phosphatidylglycerol than the inactive TH-2PLD

mutant. We demonstrated that Ala426 and Lys438 of TH-2PLD play a

role in sensing the head group of phospholipids.

Abbreviations

PA, phosphatidic acid; PC, phosphatidylcholine; PG, phosphatidylglycerol; PLD, phospholipase D; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-

phosphocholine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine];

PpNP, phosphatidyl-p-nitrophenol; RU, resonance unit; SPR, surface plasmon resonance; SUV, small unilamellar vesicle.

2672 FEBS Journal 274 (2007) 2672–2681 ª 2007 The Authors Journal compilation ª 2007 FEBS