Phospholipase D mechanism using Streptomyces PLD

Biochimica et Biophysica Acta 1791 (2009) 962–969

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Review

Phospholipase D mechanism using Streptomyces PLD Yoshiko Uesugi, Tadashi Hatanaka ⁎ Research Institute for Biological Sciences (RIBS), Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan

a r t i c l e

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Article history: Received 19 November 2008 Received in revised form 19 January 2009 Accepted 28 January 2009 Available online 4 February 2009 Keywords: Streptomyces Phospholipase D

a b s t r a c t Phospholipase D (PLD) plays various roles in important biological processes and physiological functions, including cell signaling. Streptomyces PLDs show significant sequence similarity and belong to the PLD superfamily containing two catalytic HKD motifs. These PLDs have conserved catalytic regions and are among the smallest PLD enzymes. Therefore, Streptomyces PLDs are thought to be suitable models for studying the reaction mechanism among PLDs from other sources. Furthermore, Streptomyces PLDs present advantages related to their broad substrate specificity and ease of enzyme preparation. Moreover, the tertiary structure of PLD has been elucidated only for PLD from Streptomyces sp. PMF. This article presents a review of recently reported studies of the mechanism of the catalytic reaction, substrate recognition, substrate specificity and stability of Streptomyces PLD using various protein engineering methods and surface plasmon resonance analysis. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Phospholipase D (PLD, EC 3.1.4.4) is a ubiquitous enzyme present in mammals, plants, and bacteria [1]. PLD catalyzes two reactions: hydrolysis of phospholipids to produce phosphatidic acids (PA) and a free alcohol, and transphosphatidylation of phosphatidyl groups to various phosphatidyl alcohols (Fig. 1). The PLDs are members of a superfamily that includes cardiolipin synthases, phosphatidylserine synthases, poxvirus envelope proteins, a Yersinia murine toxin, and several endonucleases [2,3]. All PLD superfamily members contain one or two copies of the conserved HxKxxxxD (where x is any amino acid), which is designated as the HKD motif [3,4]. The complete conservation of the HKD motifs among PLDs and their relatives implies a vital role of the HKD motif in the catalytic reaction, as verified by mutational studies. Substitution of amino acid residues in either HKD motif in mammalian PLD1 and yeast PLD (SPO14) renders the enzyme inactive [5]. The genes coding PLD have been cloned from various sources, from mammals to bacteria [6–8]. For comparison, the primary structures of PLDs are presented in Fig. 2. They have four conserved regions (regions I–IV) except for endonuclease from Salmonella typhimurium (Nuc) [9,10]. Both regions II and IV contain one catalytic HKD motif; some have additional conserved sequences (I and III). However, low similarity exists in the remaining parts of the molecules. The α-type PLD (PLDα) in plants possesses a calcium-binding and phospholipidbinding domain (C2 domain) at the N-terminus. Most mammalian PLDs have the Phox (PX) and the Pleckstrin (PH) homology domains

⁎ Corresponding author. Tel.: +81 866 56 9452; fax: +81 866 56 9454. E-mail address: [email protected] (T. Hatanaka). 1388-1981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2009.01.020

instead of the C2 domain in plants. The PX and PH domains also are present in yeast PLDs and ζ-type PLDs (PLDζ) from Arabidopsis [11]. Interestingly, the overall sequences of PLDζs are more similar to mammalian PLDs than to other plant PLD types. The PX and PH domains have been shown to mediate membrane targeting of the protein and are closely linked to polyphosphoinositide signaling [12]. In contrast, microbial PLDs lack such regulatory domains. Streptomyces PLDs have only four regions that are necessary for the PLD activities, and show the most compact structure among many sources. Several Streptomyces PLDs have been sequenced, for example PLD from S. antibioticus [13,14], S. cinnamoneus [15], S. halstedii [16], S. septatus [17], and Streptomyces sp. PMF strain [18,19]. These PLDs show significant sequence similarity and belong to the PLD superfamily containing two HKD motifs. A different PLD, isolated from S. chromofuscus [20,21] has very little sequence homology with the other Streptomyces PLDs [14]. This PLD contains a non-typical HKD motif. However, it has two sequences (e.g., 187HxKxxxD193 and nearby 200 HxKxxxxxxxD210) in the same region of the protein as one of the HKD motifs in the other Streptomyces PLDs. Moreover, this PLD differs from other Streptomyces PLDs in that it contains two metal ions (iron and manganese) as essential cofactors for catalytic activity [22]. Almost all Streptomyces PLDs show Ca2+-independent activity, whereas S. chromofuscus PLD, which is an iron-containing enzyme, is activated by Ca2+ [21]. This review focuses on the former Streptomyces PLDs possessing the HKD motifs. Most members of the PLD superfamily possess two HKD motifs, but the bacterial endonucleases have only one HKD motif. Stuckey and Dixon solved the tertiary structure of bacterial endonuclease, Nuc, as the first crystal structure of a member of the PLD superfamily [23]. Nuc dimerizes to perform catalysis; as shown in Fig. 3A, the interface is required for catalysis. Each subunit consists of an eight-stranded

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Fig. 1. Reactions catalyzed by PLD (A) and structure of typical phospholipid head groups as substrate (B). R represents an alkyl chain; X represents a polar head group.

mixed β-sheet that is flanked by five α-helices. Additionally, the structure of Nuc supported the importance of the histidine and lysine of HKD motif in each subunit, and revealed that the four histidine and lysine residues are clustered together in the active center of the dimeric enzyme. Leiros et al. determined the crystal structure of the PLD from Streptomyces sp. PMF (PMFPLD) [19] (Fig. 3B). PMFPLD is a monomer consisting of two domains with similar topology; PMFPLD includes 35 secondary structure elements arranged as an α–β–α–β–αsandwich structure. Two β sheets comprising nine and eight β strands are flanked by 18 α helices. Moreover, several extended loop regions are shown. One of these loop regions (residues 382–389), which is conserved in most Streptomyces species, is inferred to be an interfacial binding region. The active site region is also located at the interface between two structural domains. The entrance of the active site is cone-

shaped, with the width across the top of about 30 Å. The two HKD motifs were opposed, forming an active well. As shown in Fig. 3, the structure of PMFPLD resembles that of Nuc homodimer. Moreover, the crystal structure of human tyrosyl-DNA phosphodiesterase (Tdp1) shows that Tdp1 folds similarly to PMFPLD and Nuc homodimer [24]. Transphosphatidylation is a useful reaction synthesizing natural phospholipids, such as phosphatidylserine (PS) and phosphatidylglycerol (PG), and novel artificial phospholipids [25–27]. These phospholipids have been used for pharmaceuticals, foods, cosmetics, and other industries. Transphosphatidylation is usually carried out in a biphasic system consisting of water and water-insoluble organic solvents. The reaction is usually accompanied with various amounts of the hydrolysis product PA. The economical point of the phospholipid synthesis depends on the selectivity of the product and byproduct. Therefore, the elucidation of the catalytic reaction and the recognition of the substrate mechanism of PLD are important for understanding biological properties of PLD. The PLDs from microorganisms such as Streptomyces have been widely used for industrial phospholipid syntheses because of their higher transphosphatidylation activity than those of many other documented sources [28,29]. In addition, Streptomyces PLDs present advantages in their broad substrate specificity and the ease of enzyme preparation. The recombinant Streptomyces PLDs are secreted into the extracellular medium as an active form. Furthermore, the tertiary structure of PLD has been elucidated only for PLD from Streptomyces sp. PMF. Therefore, Streptomyces PLDs are thought to be the most suitable for studying the reaction mechanism among PLDs from other sources. For that reason, it might be applicable to the elucidation of those of other PLDs. Herein, we review recent works describing the mechanism of the catalytic reaction and substrate recognition in PLD, particularly addressing Streptomyces PLDs. 2. The catalytic mechanism

Fig. 2. Scheme of the primary structure of PLDs. The N-terminal C2 domain, the PX and PH domains, and conserved regions (I–IV) in PLDs are represented. Regions II and IV contain the two HKD motifs forming the active site. Streptomyces sp. strain PMF PLD (ExPASy primary accession number: P84147), PLDα2 from cabbage (accession number: P55939), PLD1 from human (accession number: Q13393), Saccharomyces cerevisiae SPO14 (accession number: P36126), and Streptomyces typhimurium Nuc (accession number: Q79SE0) are shown.

General findings related to the PLD catalyzed reactions are consistent with formation of a phosphatidyl-enzyme intermediate. Early evidence showing retention of configurations at the substrate phosphorous atom in the reactions catalyzed by cabbage PLD and Escherichia coli phosphatidylserine synthase suggest a “ping-pong” type of reaction through the formation of a covalent phosphatidylenzyme intermediate [30–32].

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Fig. 3. Tertiary structures of phospholipase D family members. (A) The proposed catalytically active form of Nuc is depicted as a MOLSCRIPT ribbon diagram (monomer A in yellow, monomer B in purple, and the variable loop in red). The arrows and coils represent b-strands and α-helices, respectively. The N- and C-termini are marked as N and C, respectively. The Cα atoms of the consensus sequence residues are drawn as cpk models with the following color scheme: His (green), Lys (dark blue), Asp (red), Ser (orange), and Asn (cyan). This structure is adapted from Ref. [24]. (B) The crystal structure of PMFPLD is shown with the Swiss-PDB viewer. The amino acid residues in the catalytic HKD motifs are presented respectively with the following color scheme: His (green), Lys (blue), and Asp (red). The N- and C-termini are marked respectively as N and C, respectively.

The first step is formation of a covalently linked phosphatidylenzyme intermediate through nucleophilic attack on the phosphorus atom by a His residue of one of the HKD motifs. Gottlin et al. demonstrated that the histidine residue in the HKD motif of Nuc is actually the nucleophile in the catalytic reaction via a covalent phosphohistidine intermediate [33]. The second step involves hydrolysis or transphosphatidylation of the intermediate by a water molecule or an alcohol. This two-step mechanism offers an attractive explanation for essential catalytic amino acid residues on PLD. Sung et al. reported that Ser911 of human PLD1 is an intrinsic nucleophile, which transfers a proton to the histidine residue in the C-terminal HKD motif [5]. These findings suggested a catalytic mechanism where a histidine residue of the HKD motif in one subunit acts as the nucleophile attacking the phosphate of the phosphodiester bond, and another histidine residue in the other HKD motif functions as a general acid protonating the leaving group. A similar mechanism has also been proposed for Yersinia pestis murine toxin (Ymt), another PLD superfamily member [34]. However, it remains unclear which of the two HKD motifs in PLD possesses the nucleophilic histidine residue because the subunits of Nuc are mutually indistinguishable as a result of their symmetric structure. Understanding of the catalytic mechanism has been persistently hindered for the last decade by the problem described above. To

elucidate which HKD motif has the nucleophilic property, firstly, Xie et al. and Iwasaki et al. investigated the roles of the two HKD motifs in the catalytic reaction using N-terminal and C-terminal halves of rat brain PLD1 and S. antibioticus PLD [35,36]. Based on the crystal structure of PMFPLD, a histidine residue in the N-terminal HKD motif is presumed to act as the nucleophile [37]. Leiros et al. showed that the two Asp residues in the active site (Asp202 and Asp473) formed strong hydrogen bonds to the two His residues of the HKD motifs in unliganded PMFPLD, whereas the side-chain of Asp473 formed an ion-pair interaction to His170 in PMFPLD in the presence of the substrate. They presented a hypothetical mechanism of catalytic reaction by PLD, as portrayed in Fig. 4. Raymond et al. demonstrated that His493 in C-terminal HKD motif of tyrosyl-DNA phosphodiesterase I (Tdp1) serves as a general acid during initial transesterification and that Lys495 and Asn516 also participate in the general acid reaction [38]. Furthermore, Uesugi et al. provided experimental evidence that His170 in the N-terminal HKD motif has a role of the catalytic nucleophile by surface plasmon resonance (SPR) analysis of the interaction between natural substrates and two inactive mutants [39]. They used inactive mutants, in which the His170 or His443 of the N-terminal or C-terminal HKD motifs of the PLD from Streptomyces septatus TH-2 (TH-2PLD) was substituted with Ala for evaluation of retaining a covalent phosphatidyl-enzyme intermediate (Fig. 1). In summary, previous reports present the PLDcatalyzed reaction mechanism as follows. (i) The imidazole nitrogen of His170 in the N-terminal HKD motif attacks the phosphorus atom of the substrate as a nucleophile, and His443 in the C-terminal HKD motif delivers a hydrogen to the OR′ leaving group. Subsequently, (ii) PLD and phospholipids form a phosphatidylhistidine intermediate and an alcohol is released. (iii) An alcohol or water activated by the imidazole nitrogen of His443 attacks the phosphatidyl intermediate, followed by (iv) dissociation of PLD from the intermediate and production of a new phospholipid or phosphatidic acid. This reaction mechanism via two HKD motifs might be common in PLDs from many sources because HKD motifs are conserved in mammals and bacteria, as well as in many intermediate species. 3. Substrate recognition A PLD from S. septatus TH-2 (TH-2PLD) showed the highest specific activity and the highest ratio of the transphosphatidylation activity to the hydrolytic activity among Actinomycetes PLDs [29]. Although the primary sequences have high homology (about 70%) among known Streptomyces PLDs that contain two HKD motifs, these PLDs exhibit different specific activity (7.1–90 unit/mg) in transphosphatidylation and different ratios (5.9–12.9) of the transphosphatidylation activity to the hydrolytic activity [29]. Moreover, the compositions of phosphatidic acid, which hydrolyzed from transphosphatidylated products by the side reaction, during the transphosphatidylation reaction of Streptomyces PLDs were significantly different [40]. These findings implied that other residues different from the two HKD motifs are involved in the recognition of phospholipids and affect the selectivity of the transphosphatidylation reaction. Uesugi et al. recently constructed a chimeric gene library between two highly homologous plds, which indicated different activity in transphosphatidylation, using repeat-length independent and broad spectrum (RIBS) in vivo DNA shuffling [41] to investigate the contribution of amino acid residues to the enzyme reaction of Streptomyces PLD [39] (Fig. 5). RIBS in vivo DNA shuffling is a novel method of random chimeragenesis based on highly frequent deletion formation of the introduced plasmids in the E. coli ssb-3 strain [42] and a deletion-directed chimera selection system that uses the rpsL+ gene as a reporter [41]. By comparing the activities of chimeras, it is suggested that the two regions, residues 188–203 and 425–442 of TH2PLD, are related to catalytic reactions and substrate recognition. Based on the crystal structure of PMFPLD, these regions are present in

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of PMFPLD indicated that the active site is located in the bottom of the center cleft of the molecule and also that it is covered by HKD motifs and GG/GS motif. They inferred that the GG/GS motif might control the conformation or access of the active center cleft, consequently affecting the interaction between PLD and the substrate. Very recently, Masayama et al. examined three residues Trp187, Tyr191, and Tyr385 which surround His 168 and His442 in HKD motifs of Streptomyces antibioticus PLD, and enabled it to synthesize phosphatidylinositol (PI), which cannot be produced using the wildtype Streptomyces PLDs, through site-directed and saturation mutagenesis [45]. They also demonstrated that the mutant with the above three residues substituted with Phe, Arg, and Tyr, respectively, was able to transphosphatidylate various cyclohexanols with a preference for bulkier compounds, whereas hydrolytic activity toward PC of the mutant was much lower than that of wild type. When these results are combined with the results described above by Uesugi et al., Trp187 and Tyr191 of S. antibioticus PLD correspond to Trp189 and Tyr193 of TH-2PLD, supporting that the N-terminal flexible loop region (residues 188–203 of TH-2PLD) responsible for the recognition of phospholipids.

Fig. 4. The reaction mechanism for PMFPLD on a PC substrate: R, diacylglycerol (DAG); R′, choline. The reaction that takes place when the product re-enters the active site the deadend phosphohistidine is formed and is illustrated below the horizontal line. Adapted from Ref. [37].

the two flexible loop regions between β7 and α7 (residues 188–203) and between β13 and β14 (residues 425–442) and are face-to-face in the domain–domain interface (Fig. 6). Two flexible loops, in coordination with each loop, form the entrance of the active well consisting of two HKD motifs. It is reasonable to consider that the two flexible loops are the entrance gate for phospholipid substrates from a geometrical perspective. Uesugi et al. further identified Gly188, Asp191, Ala426, and Lys438 of TH-2PLD as the key amino acid residues involved in the recognition of phospholipids [39,43]. The local environment surrounding key residues Gly188, Asp191, Ala426, and Lys438 of TH-2PLD is presented in Fig. 6. These key residues are located in two flexible loops separate from the two histidine residues of the catalytic HKD motifs. This result suggests that these residues contribute to the catalytic reactions, although they do not affect the HKD motifs directly. Residues 191 and 438 of TH-2PLD were located at the entrance of the active well. They were exposed to a solvent. On the other hand, residues 188 and 426 were located inside the enzyme. In another study, Ogino et al. recently showed that the glycine–glycine (GG) motif and glycine–serine (GS) motif, which are located seven residues downstream from each HKD motif, of PLD from Streptoverticillium cinnamoneum are key motifs that affect transphosphatidylation activity [44]. Three mutants G215S, G216S, and G216S-S489G exhibited approximately 9–27-fold increased transphosphatidylation activity toward dipalmitoyl phosphatidylcholine (PC) than wild-type. The predicted structure of Stv. cinamoneum PLD based on the crystal structure

Fig. 5. Random chimeragenesis between pldp and th-2pld genes by RIBS in vivo DNA shuffling. The random chimeragenesis (A) strategy is detailed in Ref. [41]. For construction of shuffling vector, two homologous genes (pldp and th-2pld) were placed in the same direction; a cassette containing the Gmr and E. coli rpsL+ genes was inserted between them. In fact, rpsL+ encodes the ribosomal protein S12 [41], the target of Sm. The transformation of E. coli MK1019 (ssb-3 rpsL (Smr)) with pACTIS4b (pldp/Gmr-rpsL+/ th-2pld) altered the phenotype of cells from Smr to Sms (and also Gms to Gmr) because the Sms ribosome was reconstituted with the wild-type RpsL protein encoded by the plasmid. The Gmr-rpsL+ cassette is deleted simultaneously from the plasmid and the cells reverse their phenotype from Sms/Gmr to Smr/Gms when recombination occurs between two homologous genes. Consequently, the intact form of the chimeric gene is selectable by Sm and Cm without expression. Primary structures of TH-2PLD, PLDP, and eight chimeras used for this study are illustrated schematically. (B) Specific activities of PLDs in the transphosphatidylation and hydrolytic reactions. Transphosphatidylation activities were measured at pH 5.5 with 13 mM PpNP. Hydrolytic activities were determined at pH 5.5 with 2 mM PpNP. Data are expressed as means ± SD of three independent experiments. Modified from Ref. [39].

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Fig. 6. Three-dimensional structure around the identified key residues (i.e., residues 188, 191, 426, and 438 of TH-2PLD) associated with activity. (A) (left) The overall structure of TH2PLD is shown based on the crystal structure of PMFPLD using the Swiss-PDB viewer. The key residues 188, 191, 426, and 438 of TH-2PLD are shown respectively in red, pink, light green, and blue. The amino acid residues in the catalytic HKD motifs are presented respectively in orange, green, brown, yellow, aqua, and purple. The two flexible loop regions (residues 188–203 and 425–442) are shown respectively in pale pink, and yellowish green. The N- and C-termini are marked respectively as N and C. (right) Schemes of the different substrate form of phospholipids are also portrayed. (B) The local environment around the key residues is represented. The key residues are indicated in red. The pale pink circle indicates the predicted pocket for the recognition of phospholipids.

PLD belongs to the enzymes which react with water-insoluble substrates. For most of these enzymes, PLDs are also generally more active toward aggregated substrates than free substrates. Results of previous studies show that PLD activities depend on the substrate forms. Reportedly, PLD is activated at the interface with micelle-forming substrates [46]. In contrast, PLD activity is drastically reduced toward phospholipids vesicles compared with micelle-forming and monomer substrate [47]. The geometry of the aggregated substrates is the critical factor for the catalytic reaction; nevertheless, the recognition mechanism of PLD toward the substrate form remains unclear. In the hydrolysis reaction, phospholipids exist in many different aggregated forms because of their insolubility in water [48]. Synthetic short-chain phospholipids and lysophospholipids either exist as monomers at lower concentrations below the critical micelle concentration (CMC), or they form micelles above the CMC. As the length of a fatty acid chain increases, the CMC of a particular phospholipid decreases. For example, Bian et al. reported that pure diC4PC has a CMC of about 278 mM and diC7PC has a CMC of about 1.46 mM [49]. Long-chain phospholipids do not readily form micelles, but they are soluble in mixed micelles with detergents [50]. The uncharged detergent, Triton X-100, has been widely used for this purpose [51]. Long-chain phospholipids also form vesicles. The vesicles consist of small unilamellar, large unilamellar, or multi-

lamellar forms. These structures have a bilayer organization and can serve as models of biological membranes. The packing and conformation of phospholipids in the bilayer membranes are highly dependent on the chemical and physical characteristics of the phospholipids used and on their thermotropic phase transition. The transphosphatidylation assay of PLDs is usually performed in two-phase systems consisting of water and water-immiscible organic solvents. In such systems, phospholipids are only presented in an organic phase. On the other hand, enzymes and nucleophile substrates are only at an aqueous phase. The reaction is assumed to take place at the interphase of the organic and aqueous phases. In previous kinetic studies of PLDs, most reactions were carried out in emulsion systems [52,53]. Consequently, the physical state of the substrates in the transphosphatidylation reaction differs from that in the hydrolysis reaction. The substrate specificity and sensitivity to interfaces of PMFPLD were examined using various phospholipids [47]. Although PMFPLD exhibited high activity toward mixed micelles and monomeric shortchain lipids, it showed poor activity toward vesicles forms. Uesugi et al. evaluated the effect of key amino acid residues—Gly188, Asp191, Ala426, and Lys438 of TH-2PLD—on the PLD-catalyzed activities toward phospholipid substrates in different physical states such as monomer, micelle, mixed micelle, emulsion, SUV, and MLV, as presented in Fig. 6 [43]. They found that Gly188 and Asp191 play a

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crucial role in recognizing substrate forms, whereas residues Ala426 and Lys438 enhanced transphosphatidylation and hydrolysis activities, irrespective of the substrate form. In addition, residues Ala426 and Lys438 have multiple functional roles. Substituting Ala426 and Lys438 respectively with Phe and His also led to improvement in thermostability and organic solvent tolerance, and to a change in the selectivity of transphosphatidylation activity compared with that of the original chimera [43]. 4. Substrate specificity Another factor contributing to substrate recognition is specificity toward the head group of phospholipids. In general, PLDs react with various phospholipids as substrates including PC, PG, PS, phosphatidylethanolamine, PI, lyso-PC, cardiolipin, and plasmalogens. The PLDs from mammals and poppy seedlings hydrolyze PC most efficiently among several phospholipids [54,55]. Recently, Uesugi et al. directly estimated the preference of Streptomyces PLD for the head group of

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phospholipids using SPR analysis by determining the interaction for a covalent phosphatidyl-enzyme intermediate with substituted catalytic nucleophile, His443 of TH-2PLD, with Ala (Fig. 7). The SPR analysis revealed that Streptomyces PLD interacts to a much higher degree with zwitterionic phospholipid vesicles (e.g. 1-palmitoyl-2-oleyl (PO) PC) than with anionic phospholipid vesicles (e.g. POPG and POPS) [56]: Streptomyces PLD containing HKD motifs seems to prefer zwitterionic phospholipid to anionic phospholipid similarly to other PLDs. Uesugi et al. also investigated the relation between the recognition of several phospholipids and the residues Ala426 and Lys438 by SPR analysis [56] (Figs. 6, 7). By substituting Ala426 and Lys438 of TH-2PLD with Phe and His, respectively, the inactive mutant showed a much stronger interaction with POPC, as opposed to a weaker interaction with POPG than the inactive TH-2PLD mutant. Results demonstrated that Ala426 and Lys438 of TH-2PLD play a role in sensing the head group of phospholipids. Iwasaki et al. and Xie et al. showed that PLD activity is restored when the N-terminal and C-terminal fragments of Streptomyces PLD and PLD1

Fig. 7. Interaction of PLDs and phospholipid vesicles. (A) Primary structures of wild-type TH-2PLD and its inactive mutants. The gray box shows the His residue of the C-terminal HKD motif mutated to Ala. The identified residues related to the PLD reaction are shown in black boxes. (B–J) Sensorgrams at different concentrations of inactive mutants of TH-2PLD are shown. As substrate, POPC (B, C, D), POPS (E, F, G) and POPG (H, I, J) vesicles were used. The SPR sensorgrams were obtained when TH-2(H443A) (B, E, H), TH-2-F(H443A) (C, F, I), and TH-2-FH(H443A) (D, G, J) were passed over the phospholipid vesicles at 532, 355, 236, and 158 nM, respectively (from top to bottom), at a flow rate of 20 ml/min for 5 min at 25 °C, followed by buffer at the same flow rate for 10 min. Modified from Ref. [56].

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coexist, although each fragment has only negligible activities in isolation [36,35]. Therefore, PLD is assumed to change its conformation markedly before and after binding to the substrate. Uesugi et al. showed that the tertiary structural of inactive mutants, in which Ala426 and Lys438 were substituted with Phe and Ala, changed with phospholipid binding [56]. From the findings described above, we considered that two flexible loops, in which residues Gly188, Asp191, Ala426, and Lys438 of TH-2PLD exist, form the entrance of the active well consisting of two HKD motifs, and might play a role as a trigger of conformational change when PLDs bind to the substrate. Therefore, we concluded that the PLD-catalyzed reaction mechanism is the following: residues 188 and 191 of TH-2PLD affect sensitivity to the physical state of the substrate; then the two-step reaction via HKD motifs proceeded as described in the preceding section: “The catalytic mechanism”. Residues 426 and 438 of TH-2PLD act on enhancement of activities during reaction. 5. Phospholipase D stability Transphosphatidylation is a useful reaction synthesizing rare natural and novel artificial phospholipids [25–27] for pharmaceuticals, foods, cosmetics, and other industries. The transphosphatidylation reaction is usually performed in biphasic systems that include water and waterinsoluble organic solvents. An organic solvent is necessary for dissolving the phospholipids. The PLD stability for organic solvents and high temperature is important for use as a biocatalyst in these systems. Regarding PLD stability, Hatanaka and co-workers' intensive studies obtained thermolabile and thermostable PLDs from Streptomyces [16,17]. They investigated thermostability-related residues, identifying amino acid residues 188, 346, 426, and 433 as thermostability-related residues [41,57]. By substituting a key residue of a thermolabile PLD, they also were successful in significantly enhancement of its thermostability. The key residue on thermostability of enzymes is frequently related to the tolerance against organic solvents. In fact, mutation at 346 in the thermolabile PLD from K1 and mutation at 426 and 438 in TH-2PLD showed a dramatic increase in tolerance against ethyl acetate [43,58]. These results suggest that the C-terminal flexible loop region (residues 425–442 of TH-2PLD) is responsible not only for recognition of phospholipids but also PLD stability. Moreover, another study reported that Ser489 in the GS motif also contributes to thermostability [44]. 6. Conclusions and future study This review presented an examination of investigations into catalytic reactions, substrate recognition, substrate specificity, and stability of Streptomyces PLD. The PLD reaction mechanism has been studied extensively. Results reflect that the role of the highly conserved catalytic HKD motifs in the enzyme reaction is rationalized: the N-terminal HKD motif acts as a nucleophile and the C-terminal HKD motif supports formation of a substrate–enzyme complex. Regarding substrate recognition, two flexible loops of Streptomyces PLDs, which are located as an entrance of the active well composed the two HKD motifs, play important roles. Furthermore, key amino acid residues other than those in the HKD motifs, which are related to PLD-catalyzed activities, selectivity of transphosphatidylation, and stability have been identified experimentally. The substrate recognition mechanism is not simple because of complicated factors such as the physical state of substrate and interaction to the head group of phospholipids and conformational change of PLD when interacting with the substrate. However, recent insights into molecular structure and mechanism of the substrate recognition accelerate our understanding of PLDs. The catalytic mechanism of Streptomyces PLD summarized in this review is applicable to elucidation of those of PLDs from other sources because Streptomyces PLD has the most compact structure, including conserved catalytic regions among many sources. Results of these studies have aided the understanding of PLDs in signaling in mammals and plants. Furthermore, based on the obtained information of the

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