Structure of Soybean Serine Acetyltransferase and Formation of the Cysteine Regulatory Complex as a Molecular Chaperone*

Background: Serine acetyltransferase (SAT) catalyzes the limiting step in cysteine biosynthesis. Results: Analysis of soybean SAT provides insight into catalysis and protein-protein interactions. Conclusion: Key structural features are required for catalysis and formation of a stable macromolecular complex. Significance: A new role for protein complex formation in plant cysteine biosynthesis is proposed. Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase. Formation of the cysteine regulatory complex (CRC) is a critical biochemical control feature in plant sulfur metabolism. Here we present the 1.75–3.0 Å resolution x-ray crystal structures of soybean (Glycine max) SAT (GmSAT) in apoenzyme, serine-bound, and CoA-bound forms. The GmSAT-serine and GmSAT-CoA structures provide new details on substrate interactions in the active site. The crystal structures and analysis of site-directed mutants suggest that His169 and Asp154 form a catalytic dyad for general base catalysis and that His189 may stabilize the oxyanion reaction intermediate. Glu177 helps to position Arg203 and His204 and the β1c-β2c loop for serine binding. A similar role for ionic interactions formed by Lys230 is required for CoA binding. The GmSAT structures also identify Arg253 as important for the enhanced catalytic efficiency of SAT in the CRC and suggest that movement of the residue may stabilize CoA binding in the macromolecular complex. Differences in the effect of cold on GmSAT activity in the isolated enzyme versus the enzyme in the CRC were also observed. A role for CRC formation as a molecular chaperone to maintain SAT activity in response to an environmental stress is proposed for this multienzyme complex in plants.


Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase. Formation of the cysteine regulatory complex (CRC) is a critical biochemical control feature in plant sulfur metabolism. Here we present the 1.75-3.0 Å resolution x-ray crystal structures of soybean (Glycine max) SAT (GmSAT) in apoenzyme, serine-bound, and CoA-bound forms.
The GmSAT-serine and GmSAT-CoA structures provide new details on substrate interactions in the active site. The crystal structures and analysis of site-directed mutants suggest that His 169 and Asp 154 form a catalytic dyad for general base catalysis and that His 189 may stabilize the oxyanion reaction intermediate. Glu 177 helps to position Arg 203 and His 204 and the ␤1c-␤2c loop for serine binding. A similar role for ionic interactions formed by Lys 230 is required for CoA binding. The GmSAT structures also identify Arg 253 as important for the enhanced catalytic efficiency of SAT in the CRC and suggest that movement of the residue may stabilize CoA binding in the macromolecular complex. Differences in the effect of cold on GmSAT activity in the isolated enzyme versus the enzyme in the CRC were also observed. A role for CRC formation as a molecular chaperone to maintain SAT activity in response to an environmental stress is proposed for this multienzyme complex in plants.
As a critical nutrient for plants, sulfur is required for a range of biological functions and is assimilated from the surrounding environment (1,2). In plant sulfur metabolism, cysteine synthe-sis plays a central role in fixing inorganic sulfur from the environment into the metabolic precursor for cellular thiol-containing compounds (3)(4)(5)(6). As the first step in cysteine biosynthesis ( Fig. 1), serine acetyltransferase (SAT) 3 catalyzes the formation of O-acetylserine from acetyl-CoA and serine (7)(8)(9)(10). Multiple studies suggest that SAT is the limiting step in cysteine biosynthesis (7, 10 -12). For example, in many plants, SAT activity is much lower than that of the second step in the pathway, and overexpression of SAT in plants leads to large increases in cysteine content (5,7,10). In the second step of the pathway, O-acetylserine sulfhydrylase (OASS), which is a member of the ␤-substituted alanine synthase enzyme family, catalyzes formation of cysteine from O-acetylserine and sulfide using pyridoxal-5Ј-phosphate as a cofactor (13)(14)(15)(16).
A key biochemical control feature of this pathway in plants is the association of SAT and OASS to form the cysteine regulatory complex (CRC) (17)(18)(19)(20)(21)(22)(23)(24)(25). Under high sulfur conditions, formation of the CRC enhances the catalytic efficiency of SAT for O-acetylserine production. Although assembly of the CRC inhibits OASS activity in the complex, a cellular excess of OASS maintains the formation of cysteine. In a nutrient-poor state, depletion of sulfur leads to an accumulation of O-acetylserine, which disassociates the CRC to attenuate SAT activity. Physiologically, this multienzyme assembly acts as a sensor to coordinate sulfur assimilation and modulate cysteine production in plants.
Plants typically encode multiple SAT isoforms sharing ϳ60 -80% amino acid sequence homology with specific cellular localization patterns (i.e. cytosolic, chloroplastic, and mitochondrial forms). For example, Arabidopsis thaliana (thale cress) and Glycine max (soybean) contain SAT gene families with five and eight isoforms, respectively (5, 7, 9, 26 -32). In soybean, two SAT isoforms (Glyma16g03080/GmSAT1;1 and Glyma18g08910/ * This work was supported by United States Department of Agriculture Grant GmSAT2;1) have been functionally characterized with regard to their biochemical properties and regulation of activity (31,32). GmSAT1;1 is cytosolic, and GmSAT2;1 is targeted to the cytosol and plastid. Both soybean SAT isoforms are sensitive to feedback inhibition by cysteine but to varying degrees. Only the role of GmSAT1;1 in the CRC has been examined (23). Clones of SAT from Citrullus vulgaris (watermelon), Spinacea oleracea (spinach), and Allium cepa (onion) have also been reported (8,33,34).
Comparative genomics and metabolic studies indicate that the SAT gene family is essential for plant growth and that O-acetylserine plays a role as a plant growth regulatory signal (35)(36)(37). Differential expression of SAT family members is linked to responses to cadmium exposure in A. thaliana and to nickel hyperaccumulation in Thlaspi goesingense (38 -41). Studies in soybean and A. thaliana also suggest that the cysteine biosynthetic enzymes interact with signaling systems through phosphorylation-dependent pathways as a means of coordinating sulfur, nitrogen, and carbon metabolism (32,42). Moreover, efforts aimed at improving resistance to oxidative stress and enhancing nutritional content by increasing the levels of sulfur-containing amino acids in rice, wheat, potato, and soybean have focused on engineering cysteine biosynthesis (43)(44)(45)(46)(47).
At the molecular level, the bacterial SAT are more thoroughly studied than the plant forms of the enzyme. Biochemical studies of the plant SAT are largely limited to analysis of steadystate kinetic parameters (8 -9, 26 -34). Early studies of the SAT from spinach and Salmonella typhimurium suggested a ping pong bi bi kinetic mechanism involving a possible acetyl-enzyme intermediate (48 -49); however, more recent analyses of the SAT from Escherichia coli and Haemophilus influenzae indicate either a random or an ordered mechanism, respectively (50,51). Examination of the chemical mechanism in the H. influenzae SAT implicates two histidine residues as important for a general base catalysis in which a proton is abstracted from serine for nucleophilic attack on acetyl-CoA (52)(53). Crystal structures of the SAT from various bacteria and the protozoan parasite Entamoeba histolytica reveal that these enzymes form a dimer of trimers with each monomer defined by an N-terminal ␣-helical domain and a C-terminal lefthanded ␤-helix domain, which forms the active site of the enzyme at the interface of two neighboring SAT monomers (54 -58). The plant SAT appear to function as either trimeric or hexameric proteins with oligomerization depending upon protein concentration (23,24). Although various plant SAT exhibit similar kinetic properties (8, 9, 26 -34), differences in feedback inhibition by cysteine, phosphorylation state, and formation of the CRC have been linked to the C-terminal 10 -15 residues of the enzyme (9,23,24,32,59). To date, no structural information has been reported for the plant SAT.
Here we present the crystal structures of soybean SAT (GmSAT) in the apoenzyme, serine-bound, and CoA-bound forms. Structure-guided mutagenesis identifies critical residues required for catalysis and substrate interaction in the SAT reaction mechanism and a conserved arginine residue that may change position to interact with CoA during CRC formation to enhance SAT activity. The effect of CRC formation on SAT activity under low temperature conditions may allow OASS to act as a molecular chaperone of SAT in plants when the demand for sulfur assimilation and cysteine production increases in response to low temperatures.

EXPERIMENTAL PROCEDURES
Expression Constructs and Site-directed Mutagenesis-Generation of the pET-28a-GmSAT and the pET-28a-GmSAT⌬C10 expression constructs was previously described (13,23). Site-directed mutants of GmSAT were generated using the QuikChange PCR method (Agilent Technologies).
Protein Expression and Purification-GmSAT was expressed in E. coli BL21(DE3) grown at 37°C in Terrific broth containing 50 g ml Ϫ1 kanamycin until A 600 nm ϳ0.8. After induction with 1 mM isopropyl 1-thio-␤-D-galactopyranoside, the cultures were grown overnight at 18°C. Cells were pelleted by centrifugation (10,000 ϫ g; 10 min) and suspended in 50 mM Tris (pH 8.0), 500 mM NaCl, 20 mM imidazole, 1 mM ␤-mercaptoethanol, 10% (v/v) glycerol, and 1% (v/v) Tween 20. All protein purification steps were performed at room temperature. After sonication and centrifugation (30,000 ϫ g; 30 min), the supernatant was passed over a Ni 2ϩ -NTA column equilibrated in suspension buffer. The column was then sequentially washed with buffer minus Tween 20 and with 40 mM imidazole (wash buffer) and then with wash buffer containing 20 mM O-acetylserine to remove bound E. coli OASS from GmSAT. Wash buffer with 250 mM imidazole was used to elute His-tagged protein from the column. Size exclusion chromatography of the eluted protein was performed using a Superdex-200 26/60 HiLoad column equilibrated in 10 mM Tris (pH 8.0), 50 mM NaCl, and 5 mM ␤-mercaptoethanol. Fractions containing GmSAT were concentrated to 10 -20 mg ml Ϫ1 with protein quantified by a Bradford assay using bovine serum albumin as a standard. Protein was stored at room temperature without significant loss of activity for up to 30 days. Expression and purification of GmOASS and GmSAT⌬C10 and formation of the CRC by GmSAT and GmOASS were as described previously (23).

Structure of a Plant Serine Acetyltransferase
Protein Crystallization and Structure Determination-Crystals of the apoenzyme form of GmSAT were obtained by the vapor diffusion method in 10-l hanging drops of a 1:1 mixture of protein and crystallization buffer (1.8 M ammonium phosphate, 100 mM imidazole, pH 8) at 20°C over a 0.5-ml reservoir. Crystals of the GmSAT-serine complex were obtained in the same condition with the addition of 100 mM serine. Crystals of the GmSAT-CoA complex grew in 12% ethanol, 100 mM Tris (pH 7.8), 200 mM MgCl 2 , 1 mM Tris(2-carboxyethyl)phosphine hydrochloride, and 10 mM CoA using sitting drops. All crystals were stabilized in cryoprotectant (crystallization solution and 20% glycerol) before flash freezing in liquid nitrogen. Diffraction data (100 K) were collected at beamline 19BM of the Argonne National Laboratory Advanced Photon Source with indexing, integration, and scaling performed with HKL3000 (60). The structure of the GmSAT apoenzyme form was solved by molecular replacement implemented in PHASER (61) using the structure of E. coli SAT without ligands or water molecules (55) (Protein Data Bank entry 1TD3) as a search model. Model building of the GmSAT structure was performed in COOT (62), and all refinements were performed with PHENIX (63). The final model of the GmSAT apoenzyme includes residues 14 -255 in chain A and residues 12-255 in chain B. The crystal structure of the GmSAT-serine complex was determined by the difference Fourier method using the refined wild-type structure as a starting model. In the GmSAT-serine complex, residues 13-255 are modeled in both chains of the asymmetric unit. To determine the structure of the GmSAT-CoA complex, molecular replacement implemented in PHASER used the GmSAT apoenzyme structure as a search model to find six molecules in the asymmetric unit.  Table 1. Coordinates and structure factors for the GmSAT apoenzyme (4N69), the GmSATserine complex (4N6A), and the GmSAT-CoA complex (4N6B) have been deposited in the RCSB Protein Data Bank.

RESULTS
Overall Three-dimensional Structure of GmSAT-To examine the three-dimensional structure of a plant SAT, the 1.75 Å resolution x-ray crystal structure of the apoenzyme form GmSAT was solved by molecular replacement (Table 1). There were two monomers (chains A and B) per asymmetric unit. The GmSAT monomer is divided into an N-terminal domain (residues Val 12 -Phe 151 ) consisting of eight ␣-helices and the C-terminal left-handed ␤-helix domain (Ala 152 -Val 255 ) formed by multiple ␤-strand triplets ( Fig. 2A). The overall fold of the GmSAT monomer is similar to the bacterial and Entamoeba homologs with a root mean square deviation of 1.0 -2.8 Å for C ␣ atoms over ϳ190 -230 residues (54 -58). Additional structural homology with a variety of acyltransferases, including QdtC required for synthesis of dTDP-3-acetamido-3,6-dideoxy-␣-D-glucose, perosamine N-acetyltransferase, N-acetylglucosamine-1-phosphate uridyltransferase, and PglD from the UDP-N,NЈ-diacetylbacillosamine synthesis pathway (65)(66)(67)(68), was detected using the DALI server and displayed 1.1-7.2 Å root mean square deviation of C ␣ atoms over ϳ90 -130 residues, largely in the left-handed ␤-helix domain. Crystallo- where summation is over the data used for refinement. c R free is defined the same as R cryst but was calculated using 5% of data excluded from refinement. DECEMBER 20, 2013 • VOLUME 288 • NUMBER 51 graphic symmetry of GmSAT generates a trimeric structure from each chain that adopts the dimer of trimers packing observed for the bacterial SAT structures (Fig. 2B). The 3-fold axis runs the length of the trimer and allows for extensive interaction between monomers (Fig. 2, C and E). The packing interface between trimers is formed by ␣1-␣3 of each monomer and is a mix of charged and uncharged surface regions (Fig. 2D).

Structure of a Plant Serine Acetyltransferase
Although the overall structure of GmSAT shares a common fold with the bacterial and Entamoeba enzymes, structures of GmSAT in complex with either serine or CoA provide new information about substrate interaction in these enzymes.
Structures of the GmSAT-Serine and GmSAT-CoA Complexes-Crystal structures of GmSAT with serine and CoA bound were determined to 1.8 and 3.0 Å resolution, respectively (Table 1). Both structures define the active site at the C-terminal interfaces between each monomer (Fig. 3A). Clear electron density for the ligands in the GmSAT-serine and GmSAT-CoA complexes were observed (Fig. 3, B and C).
In GmSAT, the serine binding site is located at the N-terminal side of the left-handed ␤-helix domain between the second and third ␤-strand repeats of two adjacent monomers (Fig. 4). One side of the binding site is delineated by the position of Asp 168 and His 169 of one monomer and the other by the ␤1c-␤2c loop (residues 193-205) of an adjacent monomer (Fig. 4B). This loop is mobile, because it is disordered in the GmSAT-CoA complex structure. Movement of the ␤1c-␤2c loop positions Arg 203 and His 204 for charge-charge and hydrogen bonding interactions, respectively, with the substrate. Interaction between Glu 177 and Arg 203 may help stabilize the conformation of the ␤1c-␤2c loop for serine binding. Within the serine binding site, Asp 154 hydrogen-bonds with the N␦ of His 169 , which   orients the N⑀ toward the substrate hydroxyl group. The position of this residue implicates it as critical for catalysis. The substrate amine interacts with a carboxylate oxygen of Asp 168 . The side chains of Arg 203 and His 204 form additional contacts with the serine carboxylate group.
Binding of CoA occurs along the interface of the left-handed ␤-helix domains to position the thioester group near the serine binding site and catalytic residues (Figs. 3A and 5A). In contrast to the GmSAT⅐serine complex structure, the ␤1c-␤2c loop is disordered in the GmSAT⅐CoA complex. Overall, the CoA binding site is a long groove at the interface of two adjacent monomers, which provides extensive van der Waals contacts between the protein and ligand (Fig. 5A). Specific bonding interactions are contributed largely by residues from one monomer (Fig. 5B). Lys 230 and Thr 246 interact with the pyrophosphate portion of CoA. Ala 215 and Ala 233 , along with Gly 195 of the adjacent monomer, hydrogen-bond with the pantothenate arm of CoA. An additional basic residue (Arg 253 ) is positioned within 5-7 Å of the CoA phosphate group (Fig. 5, B and C). These interactions position the thioester group of CoA in proximity to the serine binding site and two histidines (His 169 and His 189 ) that may serve catalytic roles in the SAT reaction mechanism. Of these two residues, His 169 is closer (3.6 Å) to the CoA. As modeled in the GmSAT⅐CoA complex, His 189 is 6.4 Å away from the CoA thiol, but rotation of the side chain into the active site can place the imidiazole ring ϳ3.5 Å from the thiol.
Sequence comparison of the SAT from soybean (GmSAT), A. thaliana (AtSAT), E. coli (EcSAT), H. influenzae (HiSAT), and E. histoyltica (EhSAT) reveals that residues in the serine binding site and the catalytic site are highly conserved across these organisms (Fig. 6). Amino acids in the CoA binding site are also conserved, although some sequence variation is observed between homologs. Mutations of Asp 168 , His 169 , and His 189 showed the largest effects on the steady-state kinetic parameters of GmSAT ( Table  2). Substitution of either His 169 or His 189 with an alanine led to inactive enzyme; however, the H169N and H189N mutants retained activity. The H169N mutant resulted in a 1,400-fold reduction in turnover rate compared with wild-type enzyme. In comparison, the H189N mutant displayed a 30-fold slower turnover rate and increased K m values for both substrates. Mutations of Asp 168 to alanine and asparagine decreased cata-   DECEMBER 20, 2013 • VOLUME 288 • NUMBER 51 lytic efficiency ϳ1,000and 10-fold, respectively. The threedimensional structures of GmSAT and these results suggest potential functional roles for these residues Altering residues in the serine and CoA binding sites also affected the steady-state kinetic parameters of GmSAT to varying degrees ( Table 2). Within the serine binding site, alteration of Arg 203 resulted in either a loss of activity (R203A) or up to a 500-fold decrease in k cat /K m (R203K) compared with wild-type enzyme. Mutation of His 204 , which also forms hydrogen bond interactions with the substrate, to either alanine or asparagine showed that a complete loss of the imidazole ring led to decreased turnover rates and increased K m values for serine. Substitution of a glutamine for Glu 177 , which interacts with Arg 203 to potentially stabilize the position of the ␤1c-␤2c loop over the serine binding site, also reduced catalytic efficiency 13-fold with serine.

Structure of a Plant Serine Acetyltransferase
The A215V, K230M, A233V, and T246A mutants targeted residues in the CoA binding site ( Table 2). Of these mutants, substitution of a methionine for Lys 230 resulted in a 580-fold decrease in k cat /K m for acetyl-CoA, which combined a 35-fold decrease in turnover rate and a 16-fold increase in K m for the substrate. A lesser effect was observed with the T246A mutant, which had a 6-fold increase in the K m for acetyl-CoA. The A215V and A233V mutants had modest effects on steadystate kinetic parameters. The R253A mutant was not significantly different from wild-type GmSAT with regard to steady-state kinetic parameters. Overall, the mutagenesis results suggest that critical ionic protein-ligand interactions are important for binding of both substrates in the GmSAT active site.
Role of Arg 253 in the CRC-Although the GmSAT R253A mutant displayed steady-state kinetic parameters comparable with those of wild-type enzyme, the arginine residue is conserved in the SAT from soybean, Arabidopsis, and bacteria that interact with OASS to form the CRC (Fig. 6). In the E. histolytica SAT, which does not bind with OASS, the corresponding residue is a proline. Because the disordered C-terminal tail region of SAT is essential for protein-protein interaction with OASS (13,17,(21)(22)(23)(24)(25), the position of Arg 253 on the surface of the C-terminal of SAT (Fig. 5C) led us to examine the effect of mutation on SAT activity in the CRC.
Previous work analyzing macromolecular assembly of the CRC showed that association of GmSAT and GmOASS improved the catalytic efficiency of the SAT reaction (23). Formation of the CRC with either wild-type or R253A GmSAT was confirmed by size-exclusion chromatography. As observed previously, the elution profile suggests interaction of three GmOASS dimers with a trimeric form of either GmSAT form (23). The catalytic efficiency of the CRC containing the GmSAT R253A mutant was 4-fold higher than that of the GmSAT R253A mutant alone (Table 3); however, this was significantly less than the 20-fold improvement of SAT activity observed with formation of a CRC containing wild-type SAT (Table 3). Overall, formation of the CRC with the GmSAT R253A mutant led to an 8-fold reduction in catalytic efficiency compared with that of the wild-type CRC. This result suggests that Arg 253 may

Steady-state kinetic parameters of wild-type and mutant GmSAT
Reactions were performed as described under "Experimental Procedures." All parameters are expressed as mean Ϯ S.E. for n ϭ 3. ND, activity was not detected.  change position to interact with CoA during CRC formation to enhance SAT activity. Effect of CRC Formation on Protein Stability-Association of SAT and OASS to form the CRC provides a mechanism for altering enzymatic activity of both cysteine biosynthesis enzymes (17)(18)(19)(20)(21)(22)(23)(24)(25). During protein expression and purification of GmSAT, we observed that temperature was a factor in the stability of GmSAT because the protein was inactivated by cold (0 -4°C). To examine the effects of temperature on SAT activity, full-length GmSAT, GmSAT lacking the C-terminal tail (GmSAT⌬C10), and the CRC were incubated at 4 and 25°C for 60 min. The enzymatic activities of the full-length and truncated forms of GmSAT were significantly reduced following cold treatment ( Table 4). The activity loss was reversible with recovery of ϳ90% of original activity upon return to 25°C. In comparison, association of GmSAT with GmOASS as part of the CRC protected SAT activity. Similar experiments with GmOASS showed no effect of incubation temperature on enzyme function.

DISCUSSION
SAT catalyzes the first step in the cysteine biosynthesis pathway by formation of O-acetylserine from acetyl-CoA and serine ( Fig. 1) (7-12). Subsequent reaction with metabolic sulfide leads to generation of cysteine, which is an essential compound for the synthesis of multiple sulfur-containing molecules, such as methionine, glutathione, phytochelatin heavy metal-binding peptides, iron-sulfur clusters, vitamin cofactors, and an array of specialized natural products (3,6). The crystal structures of the SAT from soybean in complex with either serine or CoA and the biochemical analysis of GmSAT site-directed mutants provide new insights into the molecular mechanism of cysteine biosynthesis in plants.
GmSAT shares a common three-dimensional fold with its homologs from bacteria and the protozoa Entamoeba and maintains a highly conserved set of active site residues for catalysis and substrate recognition (Figs. 2 and 6). The overall structure of the hexapeptide repeat that defines the left-handed ␤-helical fold of SAT and other O-acyltransferases provides a highly adaptable scaffold for tailoring of substrate specificity (65)(66)(67)(68)(69). Across the O-acyltransferase family of enzymes, oligomerization of monomers into trimeric assemblies with the length of the left-handed ␤-helical fold running along the 3-fold axis provides for substrate binding at the interfaces between monomers (65-69) (Fig. 3). In addition to their overall structural organization, the x-ray crystal structures of various O-acyltransferases show that a loop corresponding to the ␤1c-␤2c loop of GmSAT extends from the ␤-helix of one monomer to interact with a neighboring subunit to act as a cover of the active site (65)(66)(67)(68)(69). Moreover, the active sites of these enzymes share a common catalytic mechanism that relies on a histidine as the general base (65)(66)(67)(68)(69).
The reaction mechanism of the bacterial SAT has been well studied, with pH dependence and solvent deuterium isotope effect experiments suggesting that a single proton is involved in the rate-limiting step and that the acetylation of serine relies on general base catalysis (52). The structures of GmSAT (Figs. 4 and 5 and Table 2) and the steady-state kinetic analysis of sitedirected mutants suggest a similar reaction mechanism (Fig. 7). Based on the GmSAT structure, Asp 154 and His 169 are positioned to interact as a dyad that enhances the basicity of the imidazole ring. Substitution of His 169 either eliminates SAT activity (H169A) or leads to a 1,400-fold slower rate of reaction (H169N), which is consistent with the loss of a general base in the reaction. Although mutants of Asp 154 were insoluble and could not be assayed, interaction between the carboxylate side chain and His 169 would facilitate catalysis. The effects of the His 169 mutants are similar to those described for the H. influenzae SAT (53). In the proposed SAT mechanism (Fig. 7), His 169 acts as a general base to abstract a proton from the serine hydroxyl group. The activated serine side chain undergoes nucleophilic attack on the carbonyl of acetyl-CoA, which leads to formation of a tetrahedral reaction intermediate.  The combined crystallographic and mutagenesis study of GmSAT also identifies other critical active site features necessary for substrate interaction with the enzyme that have not been previously described. Comparison of the structures of GmSAT in complex with either serine or CoA suggests flexibility of the ␤1c-␤2c loop helps stabilize positioning of the active site lid (Fig. 4B and Table 2). Indeed, mutagenesis of Glu 177 reduces catalytic efficiency with serine by potentially altering the interaction with Arg 203 for substrate binding. Likewise, mutation of Arg 203 to either lysine or alanine compromises GmSAT activity by altering the kinetic parameters for serine. The position of the serine in the GmSAT structure is similar to the binding of cysteine, an inhibitor, in the bacterial SAT structures (55,56). The GmSAT-serine structure confirms the mode of cysteine inhibition as a competitor for the amino acid substrate (55)(56); however, it also explains the observed effects on acetyl-CoA by the inhibitor because binding at the serine site would lock the ␤1c-␤2c loop over the cleft between adjacent monomers to alter acetyl-CoA access to the active site.
Kinetic analysis of point mutants of residues in the CoA binding site (A215V, K230M, A233V, and T246A) implicates the interaction made between Lys 230 and the backbone phosphates of the ligand as a critical determinant for substrate binding (Table 2). In general, the SAT active site relies on key ionic protein-ligand interactions for association with both substrates.
The GmSAT-CoA complex structure also led to the discovery of Arg 253 as a mediator of CoA interaction following formation of the CRC (Table 3). Arg 253 , which is conserved in the SAT that associate with OASS to form the CRC (Fig. 6), is positioned near the CoA phosphate group (Fig. 5C) but does directly interact with the ligand in the crystal structure. Because multiple studies demonstrate the essential role of the C-terminal region of SAT for interaction with the second enzyme from the cysteine biosynthesis pathway (i.e. OASS) in the CRC (17)(18)(19)(20)(21)(22)(23)(24)(25), the location of Arg 253 on the C-terminal surface of the left-handed ␤-helix region. Mutation of Arg 253 did not significantly alter the steady-state kinetic properties of GmSAT as an isolated protein. In contrast, as part of the CRC, the R253A mutant displayed an attenuated enhancement of catalytic efficiency for the SAT reaction compared with that of wild-type SAT in the CRC (Table 3). Based on the position of Arg 253 in the GmSAT structure and kinetic analysis of wild-type and mutant SAT and CRC, we suggest that binding of OASS at the C-terminal tail of SAT shifts the arginine toward the CoA binding site to enhance interaction with the substrate and improve catalytic efficiency.
In our efforts to obtain protein for structural and functional studies of GmSAT, we observed that temperature is a critical factor in the stability of GmSAT because the protein is inactivated by cold (4°C) temperature. Interestingly, SAT activity in the CRC is largely maintained following incubation at the same temperature. Removal of the C-terminal region of GmSAT, which prevents association with GmOASS (23), does not alter cold inactivation (Table 4). Similar experiments with GmOASS showed no effect of incubation temperature on activity. Cold inactivation and/or denaturation occurs when a protein loses its folded structure as a result of temperature decreases that produce a favorable change in free energy between water and nonpolar groups that weaken hydrophobic interactions to disrupt protein structure (70 -74). These can be either global or localized structural changes. Formation of the soybean CRC alters activity of SAT and OASS in the complex, and experimental studies suggest that association with OASS may reorganize the C-terminal region of SAT (22,23). In addition to effects on reaction kinetics and inhibition, these changes may allow the CRC to function as a molecular chaperone to prevent inactivation of SAT under stress conditions, in particular cold temperature. As shown here, SAT activity is sensitive to cold (4°C), but formation of the CRC largely prevents the loss of activity. This is intriguing because multiple studies suggest that expression and activity of enzymes in plant thiol metabolism increase under cold stress. A similar protective role of CRC formation was demonstrated against feedback inhibition by cysteine (24).
Multiple metabolic and cellular changes occur at the molecular level in response to low temperature stress, including the generation of reactive oxygen species that cause cellular damage (75). Plants subjected to low temperature accumulate hydrogen peroxide and the activities of reactive oxygen species detoxification enzymes require enhanced glutathione synthesis, which places increased demands on the sulfur assimilation and cysteine synthesis pathways (75)(76)(77)(78)(79)(80)(81). Thus, CRC formation may allow OASS to act as a molecular chaperone of SAT activity in plants when the demand for sulfur assimilation and cysteine production increases in response to low temperatures.
Cold adaptation of plant proteins is not well explored, but it could be a possible mechanism for sessile organisms to tolerate this environmental stress. In general, understanding cold adaptation of enzymes has a number of potential biotechnology applications for the use of cold-active biocatalysts in food processing, cold temperature detergents, and the biosynthesis of volatile intermediates (72). A possible role for CRC formation as a molecular chaperone to maintain SAT activity, which is the rate-limiting step of cysteine synthesis, in response to an environmental stress is a new role for this multienzyme complex in plants. This is relevant for plant biotechnology because understanding the role of the CRC in maintaining SAT activity may be useful for determining which SAT and/or CRC to use for improving cold tolerance in early season crops at risk for cold damage.