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J. Biol. Chem., Vol. 283, Issue 12, 7542-7553, March 21, 2008

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Acceleration of the Substrate C Deprotonation by an Analogue of the Second Substrate Palmitoyl-CoA in Serine Palmitoyltransferase^*

Hiroko Ikushiro¹, Shigeru Fujii, Yuka Shiraiwa, and Hideyuki Hayashi²

From the Department of Biochemistry, Osaka Medical College, 2-7 Daigakumachi, Takatsuki, Osaka 569-8686 and the Laboratory of Chemistry, Kansai Medical University, Hirakata, Osaka 573-1136, Japan

Received for publication, August 17, 2007 , and in revised form, December 26, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyzes the pyridoxal 5'-phosphate (PLP)-dependent decarboxylative condensation reaction of L-serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine. The binding of L-serine alone to SPT leads to the formation of the external aldimine but does not produce a detectable amount of the quinonoid intermediate. However, the further addition of S-(2-oxoheptadecyl)-CoA, a nonreactive analogue of palmitoyl-CoA, caused the apparent accumulation of the quinonoid. NMR studies showed that the hydrogen-deuterium exchange at C of L-serine is very slow in the SPT-L-serine external aldimine complex, but the rate is 100-fold increased by the addition of S-(2-oxoheptadecyl)-CoA, showing a remarkable substrate synergism in SPT. In addition, the observation that the nonreactive palmitoyl-CoA facilitated -deprotonation indicates that the -deprotonation takes place before the Claisen-type C–C bond formation, which is consistent with the accepted mechanism of the -oxamine synthase subfamily enzymes. Structural modeling of both the SPT-L-serine external aldimine complex and SPT-L-serine–palmitoyl-CoA ternary complex suggests a mechanism in which the binding of palmitoyl-CoA to SPT induced a conformation change in the PLP-L-serine external aldimine so that the C–H bond of L-serine becomes perpendicular to the plane of the PLP-pyridine ring and is favorable for the -deprotonation. The model also proposed that the two alternative hydrogen bonding interactions of His¹⁵⁹ with L-serine and palmitoyl-CoA play an important role in the conformational change of the external aldimine. This is the unique mechanism of SPT that prevents the formation of the reactive intermediate before the binding of the second substrate.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Serine palmitoyltransferase (SPT)³ is a pyridoxal 5'-phosphate (PLP)-dependent enzyme and catalyzes a decarboxylative condensation reaction of L-serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine (KDS). This reaction is the first and rate-limiting step of the sphingolipid biosynthesis, and regulation of the SPT reaction directly influences cellular sphingolipid homeostasis (1). Therefore, the molecular mechanism of the SPT reaction has been attracting attention, since it has been shown that sphingolipid is a potent bioactive lipid mediator playing crucial roles in diverse aspects of cell structure and function (1–4).

Eukaryotic SPTs have been known to exist as membrane-bound heterodimers composed of two subunits called SPTLC1 (LCB1) (long chain base 1), which does not have a PLP-binding motif, and SPTLC2 (LCB2), which carries a lysine residue that forms the Schiff base with PLP (5–10). The identification of a new third SPT subunit, SPTLC3, and an octameric SPT structure model have been proposed (11, 12). However, further studies of the SPT reaction mechanism have not been carried out, because the instability and the hydrophobic nature of eukaryotic SPTs have precluded obtaining a sufficient amount of the enzyme for detailed observations of the reaction (13).

Recently, we isolated several SPT genes from the sphingolipid-containing bacteria, such as Sphingomonas paucimobilis, Sphingobacterium multivorum, Sphingobacterium spiritivorum, and Bdellovibrio stolpii (14–16). These bacterial SPTs show about 30% identity in the amino acid sequence with eukaryotic SPT subunit proteins and conserve the amino acid residues of eukaryotic SPTs assumed to be involved in catalysis. All of the bacterial SPTs were successfully overproduced in Escherichia coli and purified as water-soluble active homodimers. Bacterial SPTs are considered to be a prototype of the eukaryotic membrane-bound enzymes, and this system was a major breakthrough that provided the first detailed mechanistic studies of the SPT reaction. The recent elucidation of a high resolution crystal structure of the S. paucimobilis SPT is expected to boost the structural/functional analysis of the catalytic reaction of this enzyme (17).

Fig. 1 shows the proposed reaction mechanisms of SPT. The first step of the SPT reaction cycle is the binding of the amino acid substrate L-serine to the enzyme and the formation of the external aldimine (II). In a previous paper (18), we spectroscopically and kinetically analyzed this step using the purified SPT, L-serine, and its analogues. Through the study, it was found that the -carboxyl group of the substrate L-serine is important for the recognition by the enzyme, and the Michaelis complex of SPT and L-serine readily undergoes transaldimination to form the external aldimine. As generally observed for the PLP-dependent enzymes, the formation of the quinonoid intermediate (carbanion) from the external aldimine is the critical step in the reaction cycle. In the early studies using a yeast cell free system, two contradictory mechanisms for the KDS formation have been proposed: formation of the quinonoid intermediate (A-III) by decarboxylation of the substrate L-serine, followed by acylation (A) and formation of the quinonoid intermediate (B-III) by -deprotonation of L-serine, followed by acylation and subsequent decarboxylation (B) (19–21). According to the report by Krisnangkura and Sweeley (21) that deuterium was introduced into the -position of KDS during the reaction in D₂O using rat liver microsomes, mechanism B has been thought to be more appropriate. However, considering the microscopic reversibility at the equilibrium between VI and B-V, the introduction of deuterium into the C2 position of KDS is possible even if the reaction proceeds via mechanism A. Therefore, the above-mentioned deuterium exchange experiment does not deny mechanism A, and it has been elusive which mechanism operates in the formation of KDS.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Proposed reaction mechanism of SPT. The Schiff base formed between PLP and the -amino group of Lys265 of SPT is in equilibrium between the enolimine and the ketoenamine (I). Mechanism A, the quinonoid intermediate (A-III) is formed by the decarboxylation of L-serine of the external aldimine intermediate (II), followed by the acylation reaction to form the final product KDS. Mechanism B, the former quinonoid intermediate (B-III) is formed by the deprotonation of the C position of the L-serine moiety of II. KDS is produced via the later quinonoid intermediate (B-V), which is formed by the acylation and the decarboxylation of B-III. The SPT-KDS external aldimine (VI) is a common intermediate in both mechanisms. B-V can be formed in mechanism A through VI by the principle of microscopic reversibility; therefore, even if the SPT reaction proceeds via mechanism A, the proton derived from the solvent can be incorporated into the KDS.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Chemicals—Palmitoyl-CoA was obtained from Funakoshi (Tokyo, Japan). Isopropyl 1-thio-β-D-galactoside and coenzyme A were from Sigma. The PD-10 columns were from Amersham Biosciences/GE Healthcare. E. coli BL21(DE3) pLysS and plasmid pET21b were from Novagen. All other chemicals were of the highest commercially available grade.

Expression and Purification of SPT—By the PCR amplification using pET21b/SPT1_mod (18) as the template, the eight amino acid residues from Ala² to Pro⁹ of the SPT gene were deleted, and new restriction sites, NdeI and HindIII, were introduced into the gene at the translation initiation and termination sites, respectively. The mutation of His¹⁵⁹ to phenylalanine (H159F) was introduced into the SPT gene by two-step PCR using the following mutagenic primers: 5'-GACTGTACGGAATTGCATATGACCGAAGCCGCCGCTCAGCCCCACGC-3' (forward1), 5'-CCTCGACGCGACAGCTTTGCGTCGATCTATGACG-3' (forward2), 5'-CGTCATAGATCGACGCAAAGCTGTCGGCGTCGAGG-3' (reverse1), 5'-GACTGTACGGTCGACTCAGCCGATGACGCCGACCGCGCGG-3' (reverse2) (mutated bases are italicized). As the first step, the two DNA fragments encoding Met¹–Asp¹⁶⁴ and Leu¹⁵⁴–Gly⁴²⁰ of SPT were PCR-amplified using the primer pairs of forward1-reverse1 and forward2-reverse2, respectively, against pET21b/SPT1_mod. Then the full-length SPT gene having the H159F mutation and the restriction site of NdeI or HindIII at each end was synthesized by the second PCR amplification against the mixture of former PCR products using the primer pair of forward1-reverse2. Each modified SPT gene was treated with both NdeI and HindIII, purified, and ligated into pET21b. Then these recombinant plasmids were used to transform the E. coli BL21 (DE3) pLysS cells. Expression of the protein was induced with 0.1 mM isopropyl 1-thio-β-D-galactoside and continued for 6 h at 37 °C. The recombinant enzyme was purified as previously described (14). The expression level of the N-terminal deleted construct was lower than that of the full-length SPT, but the activity and the physical characteristics of the purified enzyme were not significantly changed as summarized in Table 1.

View larger version (16K): [in this window] [in a new window]   FIGURE 2. Structures of S-(2-oxoheptadecyl)-CoA (A) and palmitoyl-CoA (B). The methylene bridge between the acyl carbonyl and the CoA sulfur (arrow) makes this palmitoyl-CoA analogue resistant to nucleophilic substitution reactions.

Spectrometric Measurements—The absorption spectra of SPT were recorded by a Hitachi U-3300 spectrophotometer at 25 °C. The stopped-flow spectrophotometry was performed using an Applied Photophysics SX.18MV system (Leatherhead, UK) at 25 °C. The dead time was 2.3 ms under a gas pressure of 500 kilopascals. The time-resolved spectra were collected by the SX.18MV system equipped with a photodiode array accessory and the XScan (version 1.08) control software. The absorption changes were analyzed using the program ProK-II (Applied Photophysics). The buffer solution for the spectrometric measurements contained 50 mM HEPES-NaOH (pH 7.5), 150 mM KCl, and 0.1 mM EDTA. SPT was equilibrated with this buffer by gel filtration using a PD-10 (Sephadex G-25) column prior to the measurements.

NMR Samples and Spectral Measurements—For the NMR spectroscopy, all exchangeable hydrogens of the enzyme, the ligands, and the buffer base were replaced with deuterium. Known amounts of the buffer base were dissolved in D₂O and freeze-dried. After repeating this treatment twice, the buffer solutions were reconstituted in D₂O. L-Serine, D-serine, and S-(2-oxoheptadecyl)-CoA were also subjected to the same pretreatment. They were each dissolved in 50 mM potassium pyrophosphate buffer (D₂O, pH 7.5). SPT was equilibrated twice with deuterium-substituted buffer using a PD-10 column.

The ¹H NMR spectra were measured in Wilmad 5-mm NMR tubes kept at 23 °C by a Varian UNITY 400 spectrometer operating at 400 MHz. A flip angle of 20° with a relaxation delay time of 5 s was used. To obtain a better signal-to-noise ratio, an exponential window function for 0.2- or 0.5-Hz line-broadening factor was applied to the free induction decays of NMR measurements. Chemical shifts are expressed as ppm relative to an external standard of 3-(trimethylsilyl)propionic acid-d₄.

Structural Modeling—The modeling of S. paucimobilis SPT on the crystal structure of Rhodobacter capsulatus 5-aminolevulinic acid synthase (ALAS) complexed with succinyl-CoA (Protein Data Bank code 1BWO [PDB] ) was carried out using MOE (version 2005.06; Chemical Computing Group, Montréal, Canada) according to the manufacturer's instructions. The amino acid sequence of SPT (GenBank^TM number AB055142 [GenBank] ) was aligned to that of R. capsulatus ALAS (GenBank^TM P18709 [GenBank] ) using the BLOSUM62 substitution matrix with the gap start and gap extend penalties set to 7 and 1, respectively. The SPT sequence is slightly longer than that of ALAS, and these two sequences have 34% identity. After the alignment, the residues of SPT were fitted to the coordinates of subunits D and E of ALAS. Ten intermediates were generated using a coarse minimization, and the best intermediate was chosen for subsequent studies. The model consists of the residues Arg²²– Ala⁴¹⁵ of S. paucimobilis SPT. The N-terminal 21 and C-terminal 5 residues were not modeled. The cis peptide bonds between Gly¹⁴⁹ and Glu¹⁵⁰ of chain 2 and that between Arg³¹⁹ and Glu³²⁰ of chain 1, which were considered to be artifacts during the modeling, were changed to trans and were locally energy-minimized. The crystal structure of ALAS (subunits D and E) was superimposed on the model, and by subtracting the ALAS protein, PLP and succinyl-CoA were incorporated into the SPT model. These ligand structures were used to model the PLP-L-serine aldimine and palmitoyl-CoA, respectively. The C3–C4–C4'–N and C4'–N–C–H dihedral angles of the PLP-L-serine aldimine were set to 180 and 90°, respectively, so that the conformation resembles those of the external aldimine of most PLP enzymes that activates the -proton. The aliphatic group of palmitoyl-CoA was extended from C3 of succinyl-CoA using the Molecular Builder of MOE. Two structures, one with the PLP-L-serine aldimine and the other with the PLP-L-serine aldimine plus palmitoyl-CoA, were energy-minimized using the MMFF94x force field with all of the atoms of SPT and the ligands allowed to move.

Other Methods—The SPT activity was measured according to the previously described methods with minor modification (14). Aliquots of the reaction mixture were spotted on silica gel 60 HPTLC plate (Merck), and [3-¹⁴C]KDS was separated from L-[3-¹⁴C]serine with a solvent system of chloroform, methanol, 2 N NH₄OH (40:10:1, v/v/v). The radioactivity of each KDS spot was quantified by a BAS2500 image analyzer (Fuji Film, Tokyo, Japan), using the intensity of the authentic L-[3-¹⁴C]serine. The protein concentration during the purification procedure was determined with a BCA protein assay kit (Pierce) using bovine serum albumin as the standard. The protein concentration of the purified SPT was spectrophotometrically determined using the molar extinction coefficient of 2.83 x 10⁴ M^–1 cm^–1 at 280 nm for the PLP form of the enzyme (14). SDS-PAGE was carried out using the SDS-Tris system with a 10% polyacrylamide gel according to the procedure described by Laemmli (23).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Generation of the Quinonoid Intermediate from the External Aldimine by S-(2-Oxoheptadecyl)-CoA—Sphingomonas SPT has absorption bands at 338 and 426 nm, which arise from the enolimine and ketoenamine tautomers, respectively, of the PLP-Lys²⁶⁵ Schiff base. The addition of L-serine to SPT increased the 426 nm peak and concomitantly decreased the 338 nm peak, reflecting the transaldimination process from the PLP-Lys²⁶⁵ Schiff base to the PLP-L-serine Schiff base (i.e. the external aldimine) (Fig. 3, A and B, lines 1 and 2). No absorption was observed at around 490–500 nm, indicating that the binding of L-serine alone to SPT does not yield detectable amounts of the quinonoid intermediate. Thus, the majority of the SPT-L-serine complex exists as the external aldimine. However, when S-(2-oxoheptadecyl)-CoA, an analogue for palmitoyl-CoA, was added to the SPT-L-serine complex, a band emerged at 493 nm (Fig. 3, A and B, lines 3–7). The intensity of the 493 nm band showed a hyperbolic dependence on the concentrations of S-(2-oxoheptadecyl)-CoA (Fig. 3C), and the apparent dissociation constant (K^app_d) for S-(2-oxoheptadecyl)-CoA was determined to be 35 µM. No spectral change of SPT was detected when only S-(2-oxoheptadecyl)-CoA was added to SPT. These results indicate that the binding of the palmitoyl-CoA analogue to the external aldimine complex induces the formation of the quinonoid intermediate.

View larger version (10K): [in this window] [in a new window]   FIGURE 3. Absorption spectra of SPT in the presence of L-serine and S-(2-oxoheptadecyl)-CoA. The buffer system was 50 mM HEPES–NaOH (pH 7.5) containing 150 mM KCl and 0.1 mM EDTA. The measurements were done at 25 °C. The enzyme concentration was 5 µM. A, absorption spectra of SPT. Line 1 (dotted line), the substrate-free form of SPT. Lines 2–7 were taken in the presence of 45 mM L-serine and 0, 0.04, 0.11, 0.29, 0.70, and 1.34 mM, respectively, of S-(2-oxoheptadecyl)-CoA. B, expansion of the region from 470 to 530 nm; same numbering as in A. C, titration curve of the molar extinction coefficient at 493 nm.

Kinetic Analysis of the Spectral Transition of the SPT-L-Serine Complex upon Reaction with S-(2-Oxoheptadecyl)-CoA—In order to elucidate the process of the quinonoid formation in more detail, a transient kinetic analysis was carried out on the reaction described above. SPT (42 µM) was saturated in advance with 30 mM L-serine and reacted with S-(2-oxoheptadecyl)-CoA, and the spectral changes were followed in a stopped-flow spectrophotometer. A time-dependent increase in the 493 nm absorbance was observed (Fig. 4, A and B). The reaction was followed at the S-(2-oxoheptadecyl)-CoA concentrations of 10, 20, 40, 80, 160, and 320 µM. At each concentration, the spectral change was a nearly single exponential process. However, since the S-(2-oxoheptadecyl)-CoA concentrations were of the same order as the enzyme concentration, a numerical analysis using the Pro-KII program was necessary. The spectra generated according to the following scheme were well fit by a global analysis to the experimentally obtained time-resolved spectra,

SCHEME 1

where E represents SPT, S₁ is L-serine, ES₁ is the SPT-L-serine external aldimine complex, S₂ is S-(2-oxoheptadecyl)-CoA, ES₁S₂ is the ternary complex, and EQ₁S₂ is the SPT-L-serine quinonoid intermediate complexed with S-(2-oxoheptadecyl)-CoA. The kinetic parameters were obtained as follows: = 31.7 ± 1.1 µM, k₊₂ = 2.6 ± 4.8 s^–1, and _–2 = 64.4 ± 4.2 s^–1. That the S.D. value of k₊₂ exceeds the mean value is the result of the Levenberg-Marquardt minimization and not that of the experimental errors. The calculated absorption spectrum of ES₁S₂ was very similar to the spectrum of ES₁ and had no absorption at 493 nm (Fig. 4C, lines 1 and 2). On the other hand, the calculated absorption spectrum of EQ₁S₂ showed an intense absorption at around 493 nm with a shoulder at around 460 nm. The apparent dissociation constant (K^app_d) obtained from the static spectroscopic analysis (Fig. 3C) is related to the above kinetic parameters as follows,

(Eq. 1)

The calculated value of K^app_d using the transient kinetic parameters obtained as above was 30 µM, which was very close to the experimental value of 35 µM, thus supporting the validity of the above kinetic analysis. The apparent equilibrium constant for the ES₁S₂ EQ₁S₂ reaction, k₊₂/k_–2, is 0.04. This small value explains why the quinonoid peak in Fig. 3 is small.

View larger version (11K): [in this window] [in a new window]   FIGURE 4. Time-resolved spectra for the reaction of SPT-L-serine complex and S-(2-oxoheptadecyl)-CoA. A, time-resolved spectra for the reaction of the SPT-L-serine complex and palmitoyl-CoA at pH 7.5. The L-serine-saturated SPT (42 µM SPT and 30 mM L-serine) and 320 µM S-(2-oxoheptadecyl)-CoA were reacted in 50 mM HEPES-NaOH (pH 7.5) containing 150 mM KCl and 0.1 mM EDTA at 25 °C. The heavy dotted line represents the spectrum of SPT-L-serine complex in the absence of S-(2-oxoheptadecyl)-CoA. After the addition of S-(2-oxoheptadecyl)-CoA, spectra were taken between 1.28 and 49.92 ms at 2.56-ms intervals. B, expansion of the region from 470 to 530 nm. C, calculated absorption spectra based on the analysis of the time-resolved spectra. The solid line (line 1) shows the initial spectrum of SPT-L-serine complex (ES1). The dotted line (line 2) and dashed line (line 3) represent the spectra of ES1S2 and EQ1S2, respectively, which were obtained from the global fitting analysis of the time-resolved spectra based on the model of Scheme 1.

View larger version (14K): [in this window] [in a new window]   FIGURE 5. 1H NMR spectra of L-serine during the SPT-catalyzed hydrogen-deuterium exchange reaction at C. A, 1H NMR spectrum of 10 mM L-serine in the presence of 5 µM SPT. The interaction between a chiral -proton and two nonequivalent β-methylene protons of L-serine split the NMR peaks of the - and two β-protons into a quartet and an octet peak, respectively. B–E, 1H NMR spectra of 10 mM L-serine in the presence of both 5 µM SPT and 1.4 mM S-(2-oxoheptadecyl)-CoA. Each spectrum was recorded at 6.7 (B), 20 (C), 40 (D), and 210 min (E) after the addition of S-(2-oxoheptadecyl)-CoA. The spectrum of 1.4 mM S-(2-oxoheptadecyl)-CoA was subtracted as the background from each spectrum. The integration curve (red line) is plotted on each spectrum. Hydrogen-deuterium exchange reaction at C of L-serine by the SPT decreased the intensity of the -proton peak and changed the splitting of the β-methylene protons from octet to quartet without changing the integrated value of the β-methylene protons.

View larger version (11K): [in this window] [in a new window]   FIGURE 6. Time course of the hydrogen-deuterium exchange at the-C of L-serine and D-serine. The ratios of the integrated intensity of the -proton to that of the β-protons are plotted versus the reaction time. The lines represent theoretical fits to a single-exponential decay equation (y = A · e–kappt). Open circles, 10 mM L-serine was incubated with 5 µM SPT; open triangles, 10 mM L-serine was incubated with 5 µM SPT and 1.5 mM CoA; open squares, 10 mM L-serine was incubated with 5 µM SPT and 1.4 mM S-(2-oxoheptadecyl)-CoA; closed circles, 10 mM D-serine was incubated with 5 µM SPT; closed triangles, 10 mM D-serine was incubated with 5 µM SPT and 1.5 mM palmitoyl-CoA; closed squares, 10 mM D-serine was incubated with 5 µM SPT and 1.5 mM S-(2-oxoheptadecyl)-CoA.

(Eq. 2)

where A is the initial value of the /β ratio (y), usually around 0.485, and k_app is the apparent rate constant. The values of k_app together with the half-time (t) are summarized in Table 2. No decrease in the /β ratio was observed in the control experiments (without SPT), confirming that the -deprotonation is catalyzed by the enzyme. When L-serine was incubated with only SPT, a very slow exchange of the -proton of L-serine with t = 103 ± 6 h was observed. The exchange rate in the presence of CoA was also very slow, with t = 79.1 ± 14.0 h. On the other hand, when S-(2-oxoheptadecyl)-CoA was added to the reaction solution, t decreased to 0.75 ± 0.03 h, much shorter than the above cases.

View this table: [in this window] [in a new window]   TABLE 2 Rate of the SPT-catalyzed hydrogen-deuterium exchange of the -proton of L-serine or D-serine

SCHEME 2

where E represents SPT, S₁ is L-serine, ES₁ is the SPT-L-serine external aldimine complex, and EQ is the quinonoid intermediate. Assuming that the rate of disappearance of the -proton of L-serine is equal to the rate of the C-hydrogen abstraction from ES₁, the following differential rate equation is obtained,

(Eq. 3)

where f denotes the fraction of the C-hydrogen species in the mixture of the C-hydrogen and C-deuterium species. Solving Equation 3, we obtain the following.

(Eq. 4)

Using the values [E]_t = 5 µM, [S₁] = 10 mM, and k_app = 2.56 x 10^–4 s^–1, k₊₂ is calculated to be 0.51 s^–1 form Equation 4. This value roughly coincides with the k₊₂ value of 2.2 s^–1 obtained from the analysis of the time-resolved spectra on the SPT ternary complex formation, showing the consistency of the spectroscopic and NMR analyses described above.

Similar experiments were carried out with D-serine. The exchange of the -proton of D-serine with the solvent was slow either when incubated with SPT only (t = 150 ± 5 h), SPT and palmitoyl-CoA (t = 456 ± 99 h), or SPT and S-(2-oxoheptadecyl)-CoA (t = 1948 ± 211 h).

Reaction with the True Substrate—The reaction of the SPT-L-serine external aldimine complex with the true substrate palmitoyl-CoA was followed by ¹H NMR in the same way as the analogue. In the presence of 10 mM L-serine and 5 µM SPT, the addition of 1.5 mM palmitoyl-CoA resulted in stoichiometric decreases in the signal intensity of the β-methylene proton of L-serine (data not shown), showing that the reaction completely proceeded. The hydrogen-deuterium exchange at C, as judged from the /β ratio, was 2% for 1.5 mM palmitoyl-CoA. These low levels of exchange indicate that the rate of the C–C bond formation from the quinonoid intermediate is significantly higher than the rate of reprotonation of the quinonoid intermediate (see "Discussion").

Models for the SPT-L-Serine and the SPT-L-Serine-Palmitoyl-CoA Complexes—In order to structurally explain the activation of the -proton of the external aldimine, crystallographic analyses of the enzyme-substrate binary and ternary complexes are required. The crystal structure of the substrate-free form of S. paucimobilis SPT at 1.3 Å has been reported (17). However, the crystals of the complex of this enzyme with substrates (or analogues) have not been obtained yet. All other members of the -oxamine synthase subfamily have been successfully crystallized, and structures of a number of the enzyme-ligand complexes have been determined that include the AONS-8-amino-7-oxononanoate complex (24), KBL-2-amino-3-ketobutyrate complex (25), ALAS-glycine complex (26), and ALAS-succinyl-CoA complex (26). Therefore, based on the crystal structure of R. capsulatus ALAS complexed with succinyl-CoA, structural models for the SPT-L-serine external aldimine complex and the ternary complex of the SPT-L-serine external aldimine and palmitoyl-CoA were constructed.

View larger version (48K): [in this window] [in a new window]   FIGURE 7. Model structure of the SPT-L-serine complex (the external aldimine). Modeling of SPT on the crystal structure of ALAS from R. capsulatus was carried out using MOE. The model structure of the active site of the SPT-L-serine complex is shown as pale cyan-colored ribbons in a parallel stereo view. Shown with balls and sticks and labeled are the PLP-L-serine aldimine (yellow) and catalytic residues, His159, Lys265, and Arg390 (pale cyan). His159 formed a hydrogen bond to carboxyl oxygen derived from the substrate L-serine with a distance of 2.26 Å. The -proton of L-serine colored green is not perpendicular to the plane formed from the PLP-pyridine ring and the double bond of the Schiff base and was located 4.45 Å away from the -amino group of Lys265. Panels were generated using PyMOL version 0.99 (DeLano Scientific).

In the initial model for the binary (external aldimine) complex, the C–H bond of L-serine was perpendicular to the PLP pyridine ring (the dihedral angle C4'–N–C–H being 90°). After energy minimization, however, the carboxylate group of L-serine approached His¹⁵⁹ and formed a strong hydrogen bond/ionic interaction with N2 of His¹⁵⁹ (Fig. 7). As a result, the dihedral angle of C4'–N–C–H became 159° (149°; hereafter, the values in parenthesis are those of subunit 2). Thus, the C–H bond of L-serine deviated by 60–70° from the theoretically ideal direction for -deprotonation.

The model for the ternary complex (SPT-L-serine external aldimine plus palmitoyl-CoA) is shown in Fig. 8, A and B. Palmitoyl-CoA docks with SPT as a bent structure to insert its thioester into the active center of the enzyme. The adenosine 3',5'-bisphosphate moiety of palmitoyl-CoA is seen near the entrance of the active site, and the acyl chain moiety of palmitoyl-CoA is suitably placed into the large pocket, which is rich in hydrophobic amino acid residues. Fig. 8C shows the active site view of the ternary complex. His¹⁵⁹ forms a hydrogen bond with the acyl carbonyl O (thioester O) of palmitoyl-CoA with a distance of 2.8 Å. The carboxyl group of L-serine loses its interaction with His¹⁵⁹ and, instead, forms two hydrogen bonds with the guanidino group of Arg³⁹⁰. The C–H bond takes a perpendicular orientation to the plane formed by the PLP pyridine ring and the imine of the Schiff base, which is suitable for the -proton abstraction by Lys²⁶⁵.

Substitution of His¹⁵⁹ of SPT by Phe Resulted in Inactivation of the Enzyme—For the assessment of the role of His¹⁵⁹ in the catalysis of SPT, the residue was changed to Phe, which was chosen to conserve the aromaticity and the size of the side chain. H159F SPT was stably overexpressed and purified in the same way as the wild type enzyme. In the case of the wild-type enzyme, clear UV-visible and CD spectral changes were observed by the addition of the L-serine, indicating the external aldimine formation. For H159F SPT, although the 420 nm peak increased slightly, the 330 nm peak did not decrease, and the CD spectrum did not change. The apparent K_d value for L-serine was 11.1 mM, compared with 1.4 mM of the wild-type enzyme. These results suggest the formation of the Michaelis complex, but not the external aldimine, on the reaction with L-serine. The accumulation of the quinonoid intermediate was not detected by the further addition of S-(2-oxoheptadecyl)-CoA or palmitoyl-CoA to L-serine-saturated H159F SPT. Furthermore, KDS was not detected even after 2 µM H159F was incubated with 20 mM of L-serine and 2 mM of palmitoyl-CoA for 20 min at 25 °C. Under the same substrate concentration condition, the wild-type SPT showed the activity of v/[E]_t = 0.4 s^–1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Acceleration of the -Deprotonation of the External Aldimine by the Substrate Acyl-CoA Analogue—The results presented above clearly show that the addition of the palmitoyl-CoA analogue, S-(2-oxoheptadecyl)-CoA, to the SPT-L-serine complex causes accumulation of the quinonoid intermediate, which is not detectable in the SPT-L-serine complex (Figs. 3 and 4). ¹H NMR studies indicated that S-(2-oxoheptadecyl)-CoA accelerates the -deprotonation of the L-serine moiety in the external aldimine by more than 100-fold (Fig. 6). Apparently, this enhancement in the deprotonation rate is considered to be the cause of the accumulation of the quinonoid intermediate upon the addition of S-(2-oxoheptadecyl)-CoA. Zhang and Ferreira (27) are the first who reported the acceleration of the substrate -deprotonation by the acyl-CoA substrate; they found that the physiological substrate succinyl-CoA and its analogue acetoacetyl-CoA increased the rate of glycine proton removal 250,000- and 10-fold, respectively. The activation of the -proton of the external aldimine by the binding of the second substrate acyl-CoA may therefore be a common feature among the -oxamine synthase subfamily enzymes.

View larger version (75K): [in this window] [in a new window]   FIGURE 8. Model structure of the ternary complex of SPT, L-serine, and palmitoyl-CoA. Modeling of SPT on the crystal structure of ALAS from R. capsulatus was carried out using MOE. A, overall structure of the SPT homodimer is shown as ribbons in a parallel stereo view. One monomer is shown in pale cyan, and the other is shown in pale green. Palmitoyl-CoA and PLP-L-serine aldimine are shown in purple and yellow, respectively. The molecules in the pale green monomer are shown in light colors for distinction. B, overall view of the SPT homodimer model from another angle. Object colors are the same as in A. C, close-up view of the active site region of a monomer (the left one of A). Shown with balls and sticks are the palmitoyl-CoA, PLP-L-serine aldimine, and catalytic residues, His159, Lys265, and Arg390. His159 formed a hydrogen bond with the C2 carbonyl oxygen of palmitoyl-CoA with a distance of 2.77 Å. The carboxyl group of L-serine, in turn, forms two new hydrogen bonds with the guanidino group of Arg390 with distances of 2.36 and 2.37 Å. The -proton of L-serine (green) took a perpendicular orientation to the plane formed from the PLP-pyridine ring and the double bond of the Schiff base, suitable for the -proton abstraction by the Lys265 -amino group located at a distance of 3.14 Å. Panels were generated using PyMOL version 0.99.

Estimation of the Effect of the True Substrate Palmitoyl-CoA—The true substrate palmitoyl-CoA reacts with the quinonoid intermediate, leading to the formation of the final product, KDS and CO₂. Even a transient accumulation of the quinonoid intermediate was not observed when palmitoyl-CoA was added to the SPT-L-serine external aldimine. Therefore, it is difficult to carry out a kinetic analysis of the C deprotonation in the presence of palmitoyl-CoA. However, by combining the observation of the hydrogen-deuterium exchange of L-serine in the presence of palmitoyl-CoA and the result of the kinetic analysis of the reaction with S-(2-oxoheptadecyl)-CoA, we can gain some understanding of the reaction of the SPT-L-serine external aldimine with palmitoyl-CoA.

For the hydrogen-deuterium exchange reaction of L-serine in the presence of palmitoyl-CoA, Scheme 2 is rearranged into Scheme 3,

SCHEME 3

where SH and SD represent the hydrogen and deuterium forms of L-serine, respectively, P is palmitoyl-CoA, and K is KDS. Hydrogen removed as a proton from E·SH·P is considered to be rapidly diluted by the solvent D₂O, and, for that reason, there would be no protonation of Q to regenerate E·SH·P. The K^Ser_d value (1.4 mM) is taken from the previous study (18). The other parameters were not experimentally obtained; therefore, we made the following assumptions. The K^PalCoA_d, k₊₂, and k_–2 values were considered to be the same as the corresponding K_d, k₊₂, and k_–2 values obtained from the reaction with S-(2-oxoheptadecyl)-CoA. The kinetic deuterium isotope effect for the C deprotonation of the external aldimine was assumed to be 7.3, based on the report by Onuffer and Kirsch (33). Accordingly, the K^PalCoA_d, k^H₊₂, k^D₊₂, and k^D_–2 values were set to 40 µM, 2.2 s^–1, 0.31 s^–1, and 8.9 s^–1, respectively. Using these values and an arbitrarily set k₊₃ value and the initial concentrations of [E] = 5 µM, [SH] = 10 mM, and [P] = 1.5 mM, the time course of the concentration change of each species was simulated by Pro-KII. The result showed that [SD]/([SH] + [SD]) (i.e. the extent of the hydrogen-deuterium exchange of L-serine) is highly dependent on the k₊₃ value; thus, increasing the k₊₃ value increased [SH] and decreased the extent of the exchange. It was found that in order to explain the less than 2% exchange, k₊₃ must have a value greater than 75 s^–1. This value is much greater than the k₊₂ values (2.2 s^–1) and exceeds the value of k_–2 (62 s^–1). This indicates that the quinonoid intermediate is rapidly converted into the product and is consistent with the finding that no apparent accumulation of the quinonoid intermediate was observed for the reaction of SPT with L-serine and palmitoyl-CoA. It also indicates that the C deprotonation is the rate-limiting step of the overall reaction of SPT.

Structural Considerations Based on the Model—Although the crystal structure for the SPT-substrate complex has not been obtained so far, the high homology of SPT to other enzymes of the -oxamine synthase subfamily enabled us to construct the models for the SPT-substrate binary and ternary complexes. The models shown in Figs. 7 and 8 provided a plausible mechanism that explains why the SPT-L-serine external aldimine is essentially inert to deprotonation, whereas the binding of the second substrate palmitoyl-CoA causes instantaneous -deprotonation.

View larger version (22K): [in this window] [in a new window]   FIGURE 9. Proposed conformations of the pyridoxal aldimines of L-serine and D-serine as viewed along the C–N bond. The aldehyde carbon of PLP is shown as Cp, and the atoms of the pyridine ring are indicated by the rectangular box. In both cases of the SPT-L-serine external aldimine and the SPT-D-serine external aldimine, the C–H bond of serine is not perpendicular to the imine-pyridine conjugate system; therefore, the -deprotonation is prevented. By the binding of the second substrate palmitoyl-CoA or its analogue S-(2-oxoheptadecyl)-CoA, the configuration of L-serine changes to the geometry appropriate for activation of the -proton (top). On the other hand, in the SPT-D-serine-acyl-CoA ternary complex, the C-CH2OH bond, not the C-H bond, becomes perpendicular to the imine-pyridine plane. Therefore, the acyl-CoA binding does not promote -deprotonation of D-serine (bottom).

In the ternary complex model (Fig. 8), however, the thioester O of palmitoyl-CoA forms a stable hydrogen bond with the N2 atom of His¹⁵⁹, displacing the carboxyl group of L-serine. The L-serine carboxyl group, in turn, forms hydrogen bonds/ionic interaction with the guanidino group of Arg³⁹⁰. This interaction fixes the PLP-L-serine external aldimine to a new conformation, in which the C–H bond is almost perpendicular to the imine-pyridine plane and approaches the -amino group of Lys²⁶⁵ (schematically shown in Fig. 9A). Both factors are preferable for the -deprotonation to proceed, and this properly explains the acceleration of the -deprotonation of the external aldimine by palmitoyl-CoA.

Based on these models, the phenomenon that the exchange rate of -proton of D-serine in both binary and ternary complex was extremely slow is explained as well (Fig. 9B). Assuming that the carboxyl group of D-serine is recognized by His¹⁵⁹ in the SPT-D-serine binary complex and by Arg³⁹⁰ in the SPT-D-serine-acyl-CoA ternary complex, the C–H bond is not perpendicular to the imine-pyridine plane in either of the two complexes (Fig. 9B). These conformations are clearly unfavorable for the -deprotonation.

Importantly, Webster et al. (24) have already presented a mechanism similar to this for the reaction of AONS. They interpreted the crystal structure of the AONS-product external aldimine to imply that His¹³³, which corresponds to His¹⁵⁹ of SPT, makes a hydrogen bond/salt bridge to the carboxylate group of the alanine moiety of the external aldimine and deviates the -hydrogen from the ideal orientation, whereas the additional binding of the pimeloyl-CoA substrate would disrupt this interaction, allowing the C–H bond to be perpendicular to the imine-pyridine plane.

Comparison with Other -Oxamine Synthase Subfamily Enzymes and the Role of the Active Site Residues—The above findings suggest the critical role of His¹⁵⁹ in the regulation of the -deprotonation of L-serine during the course of the catalytic reaction of SPT. This amino acid residue is conserved among all members of the -oxamine synthase subfamily: His¹³³ in AONS, His¹³⁶ in KBL, and His¹⁴² in ALAS.

In most PLP enzymes belonging to the fold type I, which includes the -oxamine synthase subfamily, the residue that exists at the re face of the PLP-Lys internal aldimine is an aromatic amino acid, such as Trp, Tyr, and Phe. The presence of His instead of these aromatic residues may be reasonable, considering the ability of His to form hydrogen bonds with the carboxyl group of the amino acid substrate and the acyl carbonyl O of the acyl-CoA substrate and its role in the precise control of the -proton activation. Our finding that H159F SPT does not seem to form the external aldimine with L-serine and lose its catalytic activity supports the idea that His¹⁵⁹ is important as an anchoring residue of the -carboxylate of the L-serine moiety in the external aldimine. However, Trp has an N atom (N1) corresponding to N2 of His and still is not found in the -oxamine synthase subfamily enzymes. The advantage of His over Trp is that it can carry a dissociable proton at N2. This proton can activate the thioester bond by simply migrating to the acyl carbonyl O during the nucleophilic attack of the carbanion of the quinonoid intermediate (Fig. 10). It can also stabilize the carboxyl group of the amino acid substrate by neutralizing the negative charge. Since the carboxylate group is almost perpendicular to the imine-pyridine plane and is expected to be activated in the external aldimine, this interaction may be important for avoiding decarboxylation of the substrate. In fact, it has recently been reported that, if the alanine moiety of the external aldimine of AONS is replaced by trifluoroalanine, which has more electron-withdrawing substituent the external aldimine undergoes decarboxylation, indicating the potential lability of the C–COO^– bond (35). Most recently, on the mouse ALAS2 isozyme, Hunter et al. (36) reported the possibility that His²⁰⁷, which corresponds to His¹⁴² of Rhodobacter ALAS, would act as an acid catalyst at the decarboxylation step of the -amino-β-ketoadipate intermediate to form 5-aminolevrinate by carrying out the single and multiple turnover rapid scanning stopped-flow experiments.

View larger version (13K): [in this window] [in a new window]   FIGURE 10. Nucleophilic attack of the carbanion of the quinonoid intermediate to the acyl carbonyl of palmitoyl-CoA. At the re face of the PLP-Lys internal aldimine of SPT, His159 is found in the position usually occupied by aromatic amino acids in most PLP enzymes of Fold type I. His159 forms a hydrogen bond with the acyl carbonyl O of palmitoyl-CoA. This interaction polarizes the carbonyl group, and most probably, His159 acts as an acid catalyst to promote the nucleophilic attack of the quinonoid C on the palmitoyl-CoA thioester.

	FOOTNOTES

 
^* This work was supported by Grant-in-Aid for Encouragement of Young Scientists (B) 16770103 and Grant-in-Aid for Scientific Research (C) 18570114 (to H. I.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by Grant-in-Aid for Scientific Research (C) 16570125 (to H. H.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed. Tel.: 81-72-684-7291; Fax: 81-72-684-6516; E-mail: ikushiro{at}art.osaka-med.ac.jp. ² To whom correspondence may be addressed. E-mail: hayashi{at}art.osaka-med.ac.jp.

³ The abbreviations used are: SPT, serine palmitoyltransferase; PLP, pyridoxal 5'-phosphate; KDS, 3-ketodihydrosphingosine; AONS, 8-amino-7-oxononanoate synthase; KBL, 2-amino-3-ketobutyrate ligase; ALAS, 5-aminolevulinic acid synthase.

	ACKNOWLEDGMENTS

 
We thank Professor S. Kominami of Hiroshima University for valuable suggestions concerning this research. We thank T. Yano for critical reading of the manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Hanada, K. (2003) Biochim. Biophys. Acta 1632, 16–30[Medline] [Order article via Infotrieve]
2. Futerman, A. H., and Hannun, Y. A. (2004) EMBO Rep. 5, 777–782[CrossRef][Medline] [Order article via Infotrieve]
3. Munro, S. (2003) Cell 115, 377–388[CrossRef][Medline] [Order article via Infotrieve]
4. Spiegel, S., and Milstien, S. (2003) Nat. Rev. Mol. Cell Biol. 4, 397–407[CrossRef][Medline] [Order article via Infotrieve]
5. Pinto, W. J., Srinivasan, B., Shepherd, S., Schmidt, A., Dickson, R. C., and Lester, R. L. (1992) J. Bacteriol. 174, 2565–2574[Abstract/Free Full Text]
6. Buede, R., Rinker-Schaffer, C., Pinto, W. J., Lester, R. L., and Dickson, R. C. (1991) J. Bacteriol. 173, 4325–4332[Abstract/Free Full Text]
7. Nagiec, M. M., Baltisberger, J. A., Wells, G. B., Lester, R. L., and Dickson, R. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7899–7902[Abstract/Free Full Text]
8. Nagiec, M. M., Lester, R. L., and Dickson, R. C. (1996) Gene (Amst.) 177, 237–241[CrossRef][Medline] [Order article via Infotrieve]
9. Weiss, B., and Stoffel, W. (1997) Eur. J. Biochem. 249, 239–247[Medline] [Order article via Infotrieve]
10. Hanada, K., Hara, T., Nishijima, M., Kuge, O., Dickson, R. C., and Nagiec, M. M. (1997) J. Biol. Chem. 272, 32108–32114[Abstract/Free Full Text]
11. Hornemann, T., Richard, S., Rutti, M. F., Wei, Y., and von Eckardstein, A. (2006) J. Biol. Chem. 281, 37275–37281[Abstract/Free Full Text]
12. Hornemann, T., Wei, Y., and von Eckardstein, A. (2007) Biochem. J. 405, 157–164[Medline] [Order article via Infotrieve]
13. Ikushiro, H., Hayashi, H., and Kagamiyama, H. (2000) in Biochemistry and Molecular Biology of Vitamin B6 and PQQ-dependent Proteins (Iriarte, A., Kagan, H. M., and Martinez-Carrion, M., eds) pp. 251–254, Birkhäuser Verlag, Basel, Switzerland
14. Ikushiro, H., Hayashi, H., and Kagamiyama, H. (2001) J. Biol. Chem. 276, 18249–18256[Abstract/Free Full Text]
15. Ikushiro, H., Hayashi, H., and Kagamiyama, H. (2003) Biochim. Biophys. Acta 1647, 116–120[Medline] [Order article via Infotrieve]
16. Ikushiro, H., Islam, M. M., Tojo, H., and Hayashi, H. (2007) J. Bacteriol. 189, 5749–5761[Abstract/Free Full Text]
17. Yard, B. A., Carter, L. G., Johnson, K. A., Overton, I. M., Dorward, M., Liu, H., McMahon, S. A., Oke, M., Puech, D., Barton, G. J., Naismith, J. H., and Campopiano, D. J. (2007) J. Mol. Biol. 370, 870–886[CrossRef][Medline] [Order article via Infotrieve]
18. Ikushiro, H., Hayashi, H., and Kagamiyama, H. (2004) Biochemistry 43, 1082–1092[CrossRef][Medline] [Order article via Infotrieve]
19. Braun, P. E., and Snell, E. E. (1968) J. Biol. Chem. 243, 3775–3783[Abstract/Free Full Text]
20. Karlsson, K. A. (1970) Chem. Phys. Lipids 5, 6–43[CrossRef][Medline] [Order article via Infotrieve]
21. Krisnangkura, K., and Sweeley, C. C. (1976) J. Biol. Chem. 251, 1597–1602[Abstract/Free Full Text]
22. Rudnick, D. A., McWherter, C. A., Rocque, W. J., Lennon, P. J., Getman, D. P., and Gordon, J. I. (1991) J. Biol. Chem. 266, 9732–9739[Abstract/Free Full Text]
23. Laemmli, U. K. (1970) Nature 227, 680–685[CrossRef][Medline] [Order article via Infotrieve]
24. Webster, S. P., Alexeev, D., Campopiano, D. J., Watt, R. M., Alexeeva, M., Sawyer, L., and Baxter, R. L. (2000) Biochemistry 39, 516–528[CrossRef][Medline] [Order article via Infotrieve]
25. Schmidt, A., Sivaraman, J., Li, Y., Larocque, R., Barbosa, J. A., Smith, C., Matte, A., Schrag, J. D., and Cygler, M. (2001) Biochemistry 40, 5151–5160[CrossRef][Medline] [Order article via Infotrieve]
26. Astner, I., Schulze, J. O., van den Heuvel, J., Jahn, D., Schubert, W. D., and Heinz, D. W. (2005) EMBO J. 24, 3166–3177[CrossRef][Medline] [Order article via Infotrieve]
27. Zhang, J., and Ferreira, G. C. (2002) J. Biol. Chem. 277, 44660–44669[Abstract/Free Full Text]
28. Hayashi, H. (1995) J. Biochem. (Tokyo) 118, 463–473[Abstract/Free Full Text]
29. Eliot, A. C., and Kirsch, J. F. (2004) Annu. Rev. Biochem. 73, 383–415[CrossRef][Medline] [Order article via Infotrieve]
30. Ploux, O., and Marquet, A. (1996) Eur. J. Biochem. 236, 301–308[Medline] [Order article via Infotrieve]
31. Hunter, G. A., and Ferreira, G. C. (1999) Biochemistry 38, 3711–3718[CrossRef][Medline] [Order article via Infotrieve]
32. Bashir, Q., Rashid, N., and Akhter, M. (2006) Chem. Commun. (Camb.) 48, 5065–5067[Medline] [Order article via Infotrieve]
33. Onuffer, J. J., and Kirsch, J. F. (1994) Protein Eng. 7, 413–424[Abstract/Free Full Text]
34. Dunathan, H. C. (1966) Proc. Natl. Acad. Sci. U. S. A. 55, 712–716[Free Full Text]
35. Alexeev, D., Baxter, R. L., Campopiano, D. J., Kerbarh, O., Sawyer, L., Tomczyk, N., Watt, R., and Webster, S. P. (2006) Org. Biomol. Chem. 4, 1209–1212[CrossRef][Medline] [Order article via Infotrieve]
36. Hunter, G. A., Zhang, J., and Ferreira, G. C. (2007) J. Biol. Chem. 282, 23025–23035[Abstract/Free Full Text]

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