PEDS Advance Access originally published online on November 5, 2008
Protein Engineering Design and Selection 2008 21(12):737-744; doi:10.1093/protein/gzn057
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Importance of the Ca2+-binding sites in the N-catalytic domain of a family I.3 lipase for activity and stability
1Department of Material and Life Science 2Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan 3CREST, JST, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
4 To whom correspondence should be addressed. E-mail: kanaya{at}mls.eng.osaka-u.ac.jp
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Abstract |
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A family I.3 lipase from Pseudomonas sp. MIS38 (PML) contains three Ca2+-binding sites (Ca1–Ca3) in the N-catalytic domain. Of them, the Ca1 site is formed only in an open conformation. To analyze the role of these Ca2+-binding sites, three mutant proteins D157A-PML, D275A-PML and D337A-PML, which are designed to remove the Ca1, Ca2 and Ca3 sites, respectively, were constructed. Of them, the crystal structures of D157A-PML and D337A-PML in a closed conformation were determined. Both structures are nearly identical to that of the wild-type protein, except that the Ca3 site is missing in the D337A-PML structure. D157A-PML was as stable as the wild-type protein. Nevertheless, it exhibited little lipase and very weak esterase activities. D275A-PML was less stable than the wild-type protein by approximately 5°C in T1/2. It exhibited weak but significant lipase and esterase activities when compared with the wild-type protein. D337A-PML was also less stable than the wild-type protein by approximately 5°C in T1/2 but was fully active. These results suggest that the Ca1 site is required to make the active site fully open by anchoring lid 1. The Ca2 and Ca3 sites contribute to the stabilization of PML. The Ca2 site is also required to make PML fully active.
Keywords: Ca2+-binding site/crystal structure/family I.3 lipase/Pseudomonas sp. MIS38/site-directed mutagenesis
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Introduction |
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Lipases (EC 3.1.1.3 [EC] ) are enzymes that hydrolyze the carboxyl ester bonds in acyl-glycerides. Lipases also possess the ability to carry out various synthesis reactions in micro-aqueous environment and often with high regio- and enantioselectivity, rendering them important tools in biotechnology (Jaeger et al., 1999
). Lipases prefer long-chain acyl-glycerides and are usually inactive under aqueous condition. Lipases become active as a result of conformational change upon contact with the interface of micelle-forming water-insoluble substrate, a phenomenon known as interfacial activation (Verger, 1997). Structural studies on several lipases have shown that the lipase activation is caused by the opening of an 
-helical lid structure that initially covers the active site (Brady et al., 1990; Brzozowski et al., 1991).
Bacterial lipases are classified into eight families (I–VIII) based on the difference in amino acid sequences and biological properties (Arpigny and Jaeger, 1999). Among them, family I, the largest group, is further classified into seven subfamilies (I.1–I.7) with the first three subfamilies comprising of Gram-negative bacterial true lipases. Family I.1 and I.2 lipases share relatively high amino acid sequence identities to each other (>30%) and are secreted via the type II secretion system (T2SS) (Arpigny and Jaeger, 1999; Filloux, 2001). In contrast, family I.3 lipases show poor amino acid sequence identities to family I.1 and I.2 lipases (<20%) and are secreted via the type I secretion system (T1SS) (Holland et al., 2005; Angkawidjaja and Kanaya, 2006). Proteins that are secreted via the T1SS usually have a C-terminal secretion signal and several repeats of a GGxGxDxux (u: hydrophobic residue) upstream of the secretion signal, termed the RTX (repeat in toxin) motif (Felmlee and Welch, 1988; Omori et al., 2001; Holland et al., 2005; Angkawidjaja and Kanaya, 2006). These repeats bind Ca2+ ions, forming a β-roll motif, initially shown by the crystal structure of P. aeruginosa alkaline protease (Baumann et al., 1993). The first six residues form a Ca2+-binding loop and the last three form a short β-strand.
A family I.3 lipase from Pseudomonas sp. MIS38 (PML) consists of 617 amino acid residues and two domains (Amada et al., 2000). The N-catalytic domain (residues 1–370) contains the active-site residues, Ser207, Asp255 and His313, as revealed by site-directed mutagenesis (Amada et al., 2000; Kwon et al., 2000). The C-domain contains several repeats of the RTX motif and a putative secretion signal near the C-terminus. PML undergoes interfacial activation (Amada et al., 2000), and requires Ca2+ for activity (Amada et al., 2000) and folding (Amada et al., 2001). The RTX motif of PML has been proposed to function as an intramolecular chaperone (Angkawidjaja and Kanaya, 2006), as deletion (Kwon et al., 2002) or mutation (Angkawidjaja et al., 2005) of this motif generates inactive proteins, which are incompletely folded. The C-domain of PML can be used as a secretion tag for extracellular production of a heterologous protein via the T1SS (Angkawidjaja et al., 2006).
The crystal structures of PML in a closed conformation (PDB code 2Z8X) (Angkawidjaja et al., 2007b) and a family I.3 lipase from Serratia marcescens (SML) in an open conformation (PDB code 2QUA) (Meier et al., 2007) have recently been determined. SML consists of 613 amino acid residues and has a 61% amino acid sequence identity to PML. These structures, which consist of the N-catalytic domain and C-terminal β-roll sandwich domain, are nearly identical with each other, except for the structures of two lids, termed lid 1 and lid 2, and the Ca2+-binding sites. According to these structures, the N-catalytic domain of PML contains the Ca2 and Ca3 sites, while that of SML contains the Ca1 and Ca2 sites. The Ca3 site is missing in the SML structure, probably because one of the aspartate residues forming this site in PML (Asp283) is replaced by Asn (Asn284) in SML. The Ca1 site is missing in the PML structure, because lid 1, which corresponds to the well-known lid of lipases, assumes a closed conformation. In the SML structure, two aspartate residues (Asp153 and Asp157) in lid 1 coordinate with the Ca2+ ion bound to the Ca1 site. These residues greatly change their positions such that they cannot coordinate with this Ca2+ ion, when lid 1 assumes a closed conformation. Based on these results, it has been proposed that this Ca2+ ion is required to stabilize an open conformation of lid 1 by anchoring it and therefore is required for activity (Meier et al., 2007; Angkawidjaja et al., 2007b). However, it remains to be determined whether the enzyme does not exhibit activity when this site is removed. In addition, the roles of other Ca2+-binding sites remain to be understood.
In this report, we constructed three mutant proteins of PML, which are designed to remove one of the Ca1–Ca3 sites, and analyzed their activities and stabilities. We also determined the crystal structures of two of these mutant proteins. Based on these results, the roles of the Ca1–Ca3 sites are discussed.
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Materials and Methods |
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Mutagenesis
The genes encoding the mutant proteins D157A-, D275A- and D337A-PMLs were amplified by PCR using the overlap extension method (Horton et al., 1990). The mutagenic primers were designed such that the codons for Asp157 (GAC), Asp275 (GAC) and Asp337 (GAC) are changed to GCA, GCC and GCC for Ala, respectively. Plasmid pUC-PML for secretion of PML (Kwon et al., 2002) was used as a template. The plasmid pUC18 derivatives for overproduction of the mutant proteins of PML were constructed as described for pUC-PML (Kwon et al., 2002). PCR was performed with 2720 Thermal Cycler (Applied Biosystems) using KOD DNA polymerase (Toyobo) according to the procedures recommended by the supplier. All DNA oligomers for PCR were synthesized by Hokkaido System Science. The DNA sequence was confirmed with an ABI Prism 310 DNA sequencer (Perkin Elmer).
For secretion of the mutant proteins of PML, Escherichia coli DH5 [F–, hsdR17(rK–, mK+), recA1, endA1, deoR, thi–1, supE44, gyrA96, relA1] was used as a host strain. The E.coli DH5 cells were first transformed with plasmid pYBCD20 harboring the lipBCD gene from Serratia marcescens (Kawai et al., 1998), and then with the pUC18 derivatives for secretion of the mutant proteins of PML. The lipBCD genes encode T1SS for SML. The resultant recombinant cells were grown in LB medium containing 50 µg/ml ampicillin and 30 µg/ml chloramphenicol at 30°C for 24 h with constant shaking. The culture was then centrifuged at 17 000 xg for 10 min at 4°C to separate the cells and supernatant fractions. The supernatant fraction was subjected to SDS–PAGE using 12% polyacrylamide gel (Laemmli, 1970), and the amount of secreted protein was estimated from the intensity of the band visualized with the Coomassie Brilliant Blue staining.
The secreted PML mutant proteins were purified as described for PML (Angkawidjaja et al., 2007a). The protein concentration was determined from UV absorption on the basis that the absorbance of a 0.1% solution (1 mg/ml) at 280 nm is 1.14 for PML and its mutant proteins. This value was calculated using an 
of 1490 M–1 cm–1 for Tyr and of 5500 M–1 cm–1 for Trp at 280 nm (Pace et al., 1995).
The circular dichroism (CD) spectra were measured on a J-725 spectropolarimeter (Japan Spectroscopic) at 25°C. The proteins were dissolved in 25 mM Tris–HCl (pH 7.5) containing 10 mM CaCl2. The protein concentration and optical path length were 0.1 mg/ml and 2 mm for far-UV CD spectra, and 0.5 mg/ml and 10 mm for near-UV CD spectra, respectively. The mean residue ellipticity, 
, which has the units of deg cm2 dmol–1, was calculated by using an average amino acid molecular weight of 110.
The esterase and lipase activities were determined in 25 mM Tris–HCl (pH 7.5) containing 10 mM CaCl2 at 30°C using 0.5 mM p-nitrophenyl laurate (C12) and 3.7% (v/v) olive oil as a substrate, respectively, as described previously (Amada et al., 2000). One unit is defined as the amount of enzyme liberating 1 µmol of fatty acid or p-nitrophenol per minute. The specific activity is defined as unit per milligram protein.
Thermal denaturation curves of the proteins were obtained by plotting the change in CD values at 220 nm against increasing temperature. The protein was dissolved in 25 mM Tris–HCl (pH 7.5) in the absence or presence of 10 mM CaCl2. The protein concentration and optical path length were 0.1 mg/ml and 2 mm, respectively. The linear rate for temperature increase was approximately 1.0°C/min. The thermal denaturation processes of all proteins examined were irreversible under this condition. The thermal denaturation curves were normalized, assuming a linear temperature dependence of the base lines of native and denatured states. The temperature, T1/2, at which 50% of the original secondary structure is lost, was estimated from the resultant normalized curves.
Crystallization, X-ray diffraction data collection and structure determination
D157A-PML and D337A-PML were concentrated to 10 mg/ml using ultrafiltration system Microcon YM-10 (Millipore), and crystallized by hanging drop vapor diffusion method as described for PML (Angkawidjaja et al., 2007a). The crystal was soaked to cryo-buffer [0.1 M 2-morpholinoethanesulfonic acid (pH 6.0) containing 10% (w/v) polyethylene glycol 20 K, 0.2 M calcium acetate, 5 mM zinc acetate and 20% (v/v) ethylene glycol] prior to flash-freezing in a nitrogen-gas stream at –173°C. X-ray diffraction data sets were collected at a wavelength of 1.0 Å on beam line BL38B1 at SPring-8, Japan. All data sets were indexed, integrated and scaled using the HKL2000 program suite (Otwinowski and Minor, 1997).
The structure was solved by the molecular replacement method using MOLREP (Vagin and Teplyakov, 1997) in the CCP4 program suite. The 1.5 Å structure of PML (Protein Data Bank entry 2Z8X) was used as a starting model. Refinement of the structures was carried out automatically using the program REFMAC (Murshudov et al., 1997) in the CCP4 program suite and manually using the program COOT (Emsley and Cowtan, 2004). Progress in structural refinement was evaluated at each stage by the free R-factor and by inspection of stereochemical parameters calculated by the program PROCHECK (Laskowski et al., 1993). The Ramachandran plot produced by PROCHECK showed that all of the residues in the structure fall in the most favored and allowed regions. The statistics for data collection and refinement are summarized in Table I. The figures were prepared by PyMol (http://pymol.sourceforge.net/).
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Protein data bank accession number
The coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 2ZJ7 for D157A-PML and 2ZJ6 for D337A-PML.
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Preparation of mutant proteins
To analyze the role of the Ca2+-binding sites (Ca1–Ca3) in the N-catalytic domain of PML, we constructed three single mutant proteins D157A-, D275A- and D337A-PMLs, in which Asp157, Asp275 and Asp337 are replaced by Ala, respectively. At the Ca1, Ca2 and Ca3 sites, respectively, the side chains of Asp157, Asp275 and Asp337 provide bidentate coordination with the Ca2+ ions. These aspartate residues are fully conserved in various family I.3 lipase sequences. Therefore, these mutations are expected to abolish the Ca2+-binding abilities of the Ca1–Ca3 sites.
All three mutant proteins were secreted from the E.coli DH5 cells into the external medium by co-expression with the T1SS for SML (Lip system) and purified to give a single band on SDS–PAGE gel (data not shown). The secretion levels varied from 15 to 30 mg/l culture and the amounts of the proteins purified from 1 l culture varied from 2 to 8 mg for different mutant proteins, both of which were comparable to those of the wild-type protein (25 mg/l culture and 5 mg) (Angkawidjaja et al., 2007a).
The far- and near-UV CD spectra of PML and its mutant proteins were measured at pH 7.5 in the presence of 10 mM CaCl2. The far-UV CD spectra of all mutant proteins were nearly identical to that of the wild-type protein (Fig. 1A). On the other hand, the near-UV CD spectrum of D275A-PML was slightly different from that of the wild-type protein, while those of D157A-PML and D337A-PML were similar to that of the wild-type protein (Fig. 1B). These results suggest that only the mutation of Asp275 to Ala causes a local conformational change, but only to a small extent.
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Crystal structures of D157A-PML and D337A-PML
In the condition used for crystallization of PML (Angkawidjaja et al., 2007a), the crystals of D157A-PML and D337A-PML were obtained, and their crystal structures were determined at 2.2 and 2.3 Å resolutions, respectively. In this condition, the crystals of D275A-PML were not obtained. The overall structures of D157A-PML and D337A-PML are essentially the same as that of the wild-type protein in a closed conformation (Fig. 2A). The root-mean-square deviations (RMSD) between the wild-type and mutant proteins, D157A-PML and D337A-PML, are 0.39 and 0.21 Å, respectively, for the coordinates of all C atoms. The crystal structures of D157A-PML around residue 157 (Fig. 2B) and D337A-PML around residue 337 (Fig. 2C) are also essentially the same as those of the wild-type protein, except that the carboxyl groups of Asp157 and Asp337 are removed in the D157A-PML and D337A-PML structures, respectively. As expected, two Ca2+ ions bind to the Ca2 and Ca3 sites of the wild-type protein and D157A-PML, while only single Ca2+ ion binds to the Ca2 site of D337A-PML. The 2Fo-Fc maps of the wild-type protein and D337A-PML around residues 337 are shown in Fig. 2D and E, respectively. These results indicate that the mutation of Asp337 to Ala eliminates the Ca2+ ion on the Ca3 site without affecting the structure of PML.
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and 8.0Enzymatic activity
The esterase and lipase activities of the wild-type and mutant proteins were determined at 30°C in the presence or absence of 10 mM CaCl2 using p-nitrophenyl laurate and olive oil as a substrate, respectively. The results are summarized in Table II. In the presence of Ca2+ ions, the specific activities of D337A-PML for both substrates were comparable to those of the wild-type protein. In contrast, D157A-PML exhibited very low esterase and little lipase activities. The specific activities of D157A-PML for p-nitrophenyl laurate and olive oil were 0.7 and <0.4% of those of the wild-type protein, respectively. Likewise, the specific activities of D275A-PML for p-nitrophenyl laurate and olive oil were greatly reduced to 3.4 and 25% of those of the wild-type protein, respectively. In the absence of Ca2+ ions, all the wild-type and mutant proteins exhibited very low esterase and little lipase activities. The specific activities of all the wild-type and mutant proteins for p-nitrophenyl laurate determined in the absence of Ca2+ ions were comparable to that of D157A-PML determined in the presence of Ca2+ ions.
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Thermal denaturation
To analyze the effect of the mutation on the stability of PML, thermal denaturation of all the wild-type and mutant proteins was analyzed by monitoring the change in CD values at 220 nm as the temperature was increased. Thermal denaturation of these proteins was irreversible at any condition examined. The thermal denaturation curves of the wild-type and mutant proteins were reproducible, unless the protein concentration, pH, and the rate of temperature increase were seriously changed. The thermal denaturation curves of the wild-type and mutant proteins measured in 25 mM Tris–HCl (pH 7.5) in the presence of 10 mM CaCl2 are shown in Fig. 3A. The thermal denaturation curves of the wild-type protein and D275A-PML measured in the absence of Ca2+ ions after dialysis against the Ca2+-free buffer are also shown in Fig. 3A. The midpoints of the transition of these thermal denaturation curves, T1/2, are summarized in Table III. As shown in Fig. 2A, eight Ca2+ ions bind to a β-roll sandwich motif in the C-terminal domain of PML. However, these internal Ca2+ ions bind to the protein too tightly to be removed by dialysis (Amada et al., 2000; Kwon et al., 2002). Therefore, it is expected that only the Ca2+ ions bound to the Ca2 and Ca3 sites are removed by dialysis.
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In the presence of Ca2+ ions, D157A-PML is as stable as the wild-type protein, while D275A-PML and D337A-PML are less stable than the wild-type protein by approximately 5°C in T1/2. However, in the absence of Ca2+ ions, D275A-PML is as stable as the wild-type protein, indicating that the mutation of Asp275 to Ala does not seriously affect the stability of PML in the absence of Ca2+ ions. Comparison of the T1/2 values of the protein determined in the presence and absence of Ca2+ ions indicates that the stabilities of the wild-type protein and D275A-PML are decreased by approximately 10 and 5°C in T1/2, respectively, upon removal of Ca2+ ions. The latter value is lower than the former one by 5°C, probably because D275A-PML contains only single Ca2+ ion at the Ca3 site in the presence of Ca2+ ions. These results suggest that the Ca2+ ions at the Ca2 and Ca3 sites equally and additively contribute to the stabilization of PML.
The near-UV CD spectrum of the wild-type protein measured in the absence of Ca2+ ions was nearly identical to that measured in the presence of 10 mM CaCl2 (data not shown), suggesting that the structure of the wild-type protein is not seriously changed upon removal of the Ca2+ ions from the Ca2 and Ca3 sites.
Thermal denaturation of PML and D157A-PML was also analyzed in the presence of 0.015% (w/v) Triton X-100 (about 0.23 mM) and either in the presence or absence of 10 mM CaCl2. The thermal denaturation curves of these proteins are shown in Fig. 3B and their T1/2 values are summarized in Table III. It has previously been reported that SML assumes an open conformation in the presence of 0.2% (w/v) Triton X-100 (Meier et al., 2007). Therefore, it is expected that PML also assumes an open conformation in this condition. However, the noises of the CD signals increase as the concentration of Triton X-100 increases and they are too large to obtain the thermal denaturation curves in the presence of 0.2% Triton X-100. Therefore, we reduced it to 0.015%. In this condition, the thermal denaturation curves of the protein was obtained, although the noises of the CD signals are still high (Fig. 3B).
The T1/2 values of PML and D157A-PML in the presence of 0.015% Triton X-100 are lower than those in the absence of it by approximately 2°C. The stabilities of these proteins are decreased in the presence of Triton X-100 when compared with those in the absence of it, probably because hydrophobic interactions that stabilize a closed conformation of lid 1 and lid 2 are reduced and conformational flexibilities of these lids increase. However, the T1/2 value of PML is higher than that of D157A-PML by 1.0°C in the presence of 0.015% Triton X-100 and 10 mM CaCl2, while it is comparable to that of D157A-PML in the presence of 0.015% Triton X-100 and absence of Ca2+ ions. These results suggest that the Ca2+ ion can bind to the Ca1 site only in a condition, in which an open conformation is induced. The contribution of this Ca2+ ion to the stabilization of PML is not so significant, probably because anchoring of lid 1 by this Ca2+ ion may not seriously affect the flexibility of lid 1.
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Discussion |
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According to the crystal structures of family I.1 (Nardini et al., 2000
) and family I.2 (Noble et al., 1993; Kim et al., 1997; Schrag et al., 1997) lipases, which show little similarity to that of PML, except for the steric configurations of the active site residues, only single Ca2+-binding site is located near the active site. Biochemical studies suggested that this site is required to make the protein fully stable (Tanaka et al., 2003) and conformation of the active site fully active (Svendsen et al., 1995; Yang et al., 2000). Studies on staphylococcal lipases, which also contain single Ca2+-binding sites but represent family I.5 lipases, also showed that these sites are required for structural stabilization and full enzymatic activity (Talon et al., 1995; Simons et al., 1996; Oh et al., 1999), but are dispensable for lipase activity (Simons et al., 1999). All the aforementioned Ca2+-binding sites are not conserved in family I.3 lipases. Namely, all three Ca2+-binding sites in the N-catalytic domain of family I.3 lipase are unique to this family. The role of each Ca2+-binding site of PML is discussed below. The Ca1 site is formed in SML in an open conformation, in which a long helix, termed lid 1, is anchored by the Ca2+ ion. This Ca2+ ion is hexacoordinated with the side chains of Gln120, Asp153 (monodentate) and Asp157 (bidentate), and main chain oxygen atoms of Thr118 and Ser144. All of these residues are conserved in PML with the same residue numbers, suggesting that the Ca1 site is formed in PML in an open conformation as well. The mutation of Asp157 to Ala neither seriously affects the lid structure of PML in a closed conformation nor the stability of PML. Nevertheless, this mutation almost fully abolishes the enzymatic activity, suggesting that this mutation prevents the formation of the Ca1 site. These hypotheses are supported by the observation that PML is stabilized by the addition of Ca2+ ions in a condition, in which an open conformation is induced, while D157A-PML is not (Fig. 3B and Table III). In the closed conformation, lid 1 is sharply bent at the middle of the helix to form a helix-turn-helix-like structure, as shown in Fig. 2A, in which the N- and C-terminal helices are termed lid 1N and lid 1C, respectively. Asp157 is located at the N-terminal region of lid 1C and fully exposed to the solvent. Upon interaction with the micellar substrate, lid 1N and lid 1C greatly shift their positions, such that one long helix is formed. The axis of this helix is almost perpendicular to the plane of Fig. 2A. According to this conformational change, Asp157 moves leftward by approximately 15 Å to form the Ca1 site. Because the Ca1 site is deeply buried inside the protein molecule, an open conformation is probably fairly unstable unless the Ca2+ ion binds to this site. Thus, the Ca1 site is required to stabilize an open conformation and to make the enzyme active.
It is noted that D157A-PML does not exhibit lipase activity, but exhibits weak esterase activity (Table II). Its specific activity for the esterase substrate is 0.7% of that of the wild-type protein. This value is very low but is significantly higher than those of the active site mutants of PML, which have been reported to be <0.06% (Amada et al., 2000; Kwon et al., 2000). PML also does not exhibit lipase activity, but exhibits weak esterase activity in the absence of Ca2+ ions (Table II). Its specific activity for the esterase substrate is similar to that of D157A-PML determined either in the presence or absence of Ca2+ ions. This esterase activity is enhanced by approximately 10 times in the presence of a nonionic detergent, such as Triton X-100 (K. Kuwahara, unpublished results). These results suggest that the Ca1 site is not required for esterase activity. In the presence of the substrate or nonionic detergent, lid 1 and lid 2 are probably dissociated from the active site. However, the active site is fully open only when lid 1 is anchored by a Ca2+ ion. Large micellar substrate of lipase can contact with the active site, only when lid 1 and lid 2 are fully open, while small substrate of esterase can contact with the active site even when these lids are partially open. This may be the reason why PML and D157-PML do not exhibit lipase activity, but exhibit weak esterase activity even in the absence of Ca2+ ions.
The Ca2+ ion at the Ca2 site is heptacoordinated by the side chains of Glu253 (monodentate), Asp275 (bidentate) and Asn284, main chain oxygen atom of Asp283 and two water molecules. It is most likely that the Ca2+-binding ability of the Ca2 site is abolished by the mutation of Asp275 to Ala, because the stability of D275A-PML is considerably decreased when compared with that of the wild-type protein in the presence of Ca2+ ions, while it is comparable to that of the wild-type protein in the absence of Ca2+ ions. The far- and near-UV CD spectra of the wild-type protein and D275A-PML suggest that the structure of PML is not seriously changed by the removal of the Ca2+ ion from the Ca2 site, but is slightly changed by this mutation. The side chain of Asp275 not only provides ligands for coordination of the Ca2+ ion but also forms hydrogen bonds with two side chain nitrogen atoms of Arg259 and the main chain nitrogen atom of Glu363. The elimination of these hydrogen bonds may cause a local conformational change. However, this conformational change is marginal, because the stability of PML in the absence of the Ca2+ ion is not seriously changed by the mutation of Asp275 to Ala (Table II).
All amino acid residues, which coordinate with the Ca2+ ion at the Ca2 site, are located in a large loop between β7-strand (Leu249-Val251) and β8-strand (Ile285-Phe288). One of the active site residues, Asp255, is also present in this loop. The enzymatic activity of PML is greatly reduced by the mutation at the Ca2 site, probably because the conformation of this loop is changed and thereby the position of Asp255 shifts from the optimal one. This conformational change is probably too small to be detected by the CD spectra. Thus, the Ca2 site is probably required to make the conformation of the active site fully active and stable.
It is noted that the β8-strand is located at the end of a long parallel β-sheet extended from one side of the first β-roll motif in the C-domain. This long parallel β-sheet appears to form a backbone of the PML structure. Formation of this long parallel β-sheet may be required to make the conformation of the active site functional, because the preceding loop of the β8-strand contains Asp255. It has been suggested that the β-roll motif acts as an intramolecular chaperon that facilitates folding of the N-catalytic domain (Angkawidjaja et al., 2007b; Meier et al., 2007). The β-roll motif may induce folding of the N-catalytic domain through the formation of this long parallel β-sheet. Because the Ca2 site stabilizes the β8-strand and its preceding loop, this site may be required to assist the chaperone-like function of the β-roll motif as well.
At Ca3 site, the Ca2+ ion is heptacoordinated by the side chains of Asp283 (monodentate) and Asp337 (bidentate), main chain oxygen atoms of Lys278 and Ala281 and two water molecules. These amino acid residues are not fully conserved in family I.3 lipases, suggesting that the Ca3 site is not conserved in these proteins. In fact, this site is missing in the SML structure. In SML, Lys278 and Asp283 are replaced by His (His279) and Asn (Asn284), respectively. The crystal structure of D337A-PML indicates that the Ca3 site is removed by the mutation of Asp337 to Ala. D337A-PML is as active as the wild-type protein, but is less stable than it by approximately 5°C in T1/2, indicating that this site contributes to the stabilization of PML. As mentioned above, the Ca2 site also contributes to the stabilization of PML to a similar extent. The Ca3 site is located relatively close to the Ca2 site, with distance around 8.4 Å. Nevertheless, both sites additively contribute to the stabilization of PML. Some family I.3 lipases may acquire the Ca3 site to increase their thermal stability.
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Funding |
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A Grant-in-Aid for Scientific Research on Priority Areas ‘Systems Genomics’ from the Ministry of Education, Culture, Sports, Science, and Technology of Japan: an Industrial Technology Research Grant Program from the New Energy and Industrial Technology Development Organization (NEDO) of Japan.
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Footnotes |
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Edited by Taiji Imoto
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Acknowledgements |
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The synchrotron radiation experiments were performed at the beam line BL38B1 in the SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal No. 2007A1336, 2007B1117). We thank Dr K. Omori for the kind gift of plasmid pYBCD20.
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References |
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Received May 20, 2008; revised September 18, 2008; accepted September 30, 2008.
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