Structural and Kinetic Analysis of Free Methionine-R-sulfoxide Reductase from Staphylococcus aureus

Free methionine-R-sulfoxide reductase (fRMsr) reduces free methionine R-sulfoxide back to methionine, but its catalytic mechanism is poorly understood. Here, we have determined the crystal structures of the reduced, substrate-bound, and oxidized forms of fRMsr from Staphylococcus aureus. Our structural and biochemical analyses suggest the catalytic mechanism of fRMsr in which Cys102 functions as the catalytic residue and Cys68 as the resolving Cys that forms a disulfide bond with Cys102. Cys78, previously thought to be a catalytic Cys, is a non-essential residue for catalytic function. Additionally, our structures provide insights into the enzyme-substrate interaction and the role of active site residues in substrate binding. Structural comparison reveals that conformational changes occur in the active site during catalysis, particularly in the loop of residues 97–106 containing the catalytic Cys102. We have also crystallized a complex between fRMsr and isopropyl alcohol, which acts as a competitive inhibitor for the enzyme. This isopropyl alcohol-bound structure helps us to understand the inhibitory mechanism of fRMsr. Our structural and enzymatic analyses suggest that a branched methyl group in alcohol seems important for competitive inhibition of the fRMsr due to its ability to bind to the active site.

Reactive oxygen species can damage cellular components, including lipids, nucleic acids, and proteins. Damage to proteins by reactive oxygen species is probably due to oxidation of side chains of amino acid residues (1). The sulfur-containing amino acids, methionine and cysteine, are the most sensitive to oxidation. Oxidation of methionine generates a diastereomeric mixture of methionine S-sulfoxide (Met-S-O) 3 and methionine R-sulfoxide (Met-R-O) (2). Methionine oxidation is associated with a variety of physiological and pathological processes, such as cellular signaling, aging, and neurodegenerative diseases (3,4). For example, methionine oxidation activates calcium/calmodulin-dependent protein kinase II in the absence of calcium (5), regulates the life span of yeast, fruit fly, and nematode (6 -8), and may advance progression of Alzheimer and Parkinson diseases (9 -12).
However, this oxidation can be reversed by the methioninesulfoxide reductases (Msrs). Two distinct families of Msrs have evolved for the stereospecific reduction of methionine sulfoxides in proteins (13,14). MsrA catalyzes the reduction of Met-S-O, whereas MsrB reduces Met-R-O. Most organisms from bacteria to humans possess a methionine sulfoxide reduction system that confers upon them the ability to repair oxidative damage and consequently impacts their longevity in oxidative environments (2,4). In addition, Msrs are involved in the virulence mechanism of some bacterial pathogens, including Mycoplasma genitalium and Neisseria gonorrhoeae (15)(16)(17). Recently, an enzyme specific for the reduction of free Met-R-O has been identified from Escherichia coli and named fRMsr (18). This protein is found in unicellular organisms, including Saccharomyces cerevisiae, but absent in multicellular organisms (19). Interestingly, fRMsr contains a GAF domain, which is a ubiquitous motif present in cyclic GMP phosphodiesterases (20). Two variants of fRMsr proteins were detected with different conserved Cys residues (19); type I fRMsrs contain three conserved Cys residues, whereas type II fRMsrs have two.
The structures and catalytic mechanisms of MsrA and MsrB are well characterized (21)(22)(23)(24). Although MsrA and MsrB are completely different in sequence and structure, they share a common catalytic mechanism involving formation of a sulfenic acid intermediate on the catalytic Cys, followed by regeneration of the oxidized catalytic Cys. Briefly, a catalytic Cys attacks the sulfur of methionine sulfoxide and forms a sulfenic acid intermediate, with concomitant release of the product, methionine. The catalytic Cys sulfenic acid then forms an intramolecular disulfide bond by interacting with a resolving Cys. The disulfide bond is reduced by reductants, and consequently the enzyme becomes active again. Thioredoxin (Trx) is generally considered the in vivo reductant, whereas dithiothreitol (DTT) can be used in vitro. In contrast, the catalytic mechanism of fRMsr is poorly understood, although previous studies suggested that its catalytic mechanism is similar to those of MsrA and MsrB, involving the common sulfenic acid chemistry. It has been found that Staphylococcus aureus, a leading cause of hospital-and community-acquired infections, contains a type I fRMsr, three MsrAs, and an MsrB (19,25). S. aureus fRMsr contains three conserved Cys residues (Cys 68 , Cys 78 , and Cys 102 ). Two crystal structures of fRMsrs from E. coli and S. cerevisae are available (Protein Data Bank codes 1VHM (18,26) and 1F5M (27), respectively). Both structures contain a disulfide bond between Cys 68 and Cys 102 (numbering is based on the S. aureus fRMsr) in the active sites, suggesting that fRMsrs use Cys residues for catalysis. The active site is enclosed in a small cavity (18,19,26,27). This enclosed cavity supports the apparent substrate specificity for free Met-R-O but not for protein-based forms. Previous studies suggested that Cys 78 functions as a catalytic residue, Cys 102 as a primary resolving Cys, and Cys 68 as a secondary resolving residue (18,19). However, the roles of these three Cys residues are unclear in the catalysis of fRMsr. Thus, the catalytic mechanism of this enzyme has yet to be elucidated.
In this study, we resolved four structural forms of the S. aureus fRMsr by x-ray crystallography: reduced form (fRMsr red ), complexed form with the substrate (fRMsr sub ), oxidized form (fRMsr ox ), and another complexed form with isopropyl alcohol (fRMsr isopro ). The first three structures represent different catalytic states of fRMsr. The last structure, fRMsr isopro , helps us to understand the inhibitory mechanism of fRMsr. We also performed biochemical analyses using the wild type S. aureus fRMsr and single and double mutants, in which the three conserved Cys are replaced with Ser. We studied the inhibitory effect of various alcohols on fRMsr. Our structural and enzymatic studies provide insights into the catalytic mechanism of fRMsr with conformational changes that occur during catalysis and into the inhibitory mechanism involving a branched methyl group of alcohols.

EXPERIMENTAL PROCEDURES
Purification, Crystallization, and X-ray Analysis-Gene cloning, protein expression, purification, and crystallization of S. aureus fRMsr have been described elsewhere for the oxidized and isopropyl alcohol-complexed forms of S. aureus fRMsr (fRMsr ox and fRMsr isopro ) (29). The crystal complexed with isopropyl alcohol was obtained from a crystallization solution consisting of 2 M ammonium sulfate and 10% (v/v) 2-propanol. For the reduced form of fRMsr (fRMsr red ), cell pellets were resuspended in ice-cold lysis buffer (20 mM Tris-HCl, pH 7.9, 500 mM NaCl, 5 mM imidazole, 10 mM ␤-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride). The purification procedures were similar to those of oxidized form, but 10 mM DTT was used in the final purification procedure of gel filtration. The crystallization condition comprised 24% polyethylene glycol 3350 and 0.35 M potassium fluoride. The substrate complex form of fRMsr (fRMsr sub ) was obtained by soaking 9 mM free Met-R-O in native crystals of mutant C68S fRMsr in which the crystallization condition comprised 26% polyethylene glycol 400 and 0.1 M MES, pH 6.4.
The crystals were soaked in a solution containing 25% (v/v) ethylene glycol used as cryoprotectant and frozen in liquid nitrogen. X-ray diffraction data were collected with an ADSC Quantum CCD 210 detector at beamlines 6C and 4A at Pohang Light Source (Pohang, South Korea). A total range of 360°was covered with 1.0°oscillation and 30-s exposure per frame. The crystal-to-detector distance was set to 150 mm. The data sets were processed and scaled using HKL 2000 (30). The fRMsr red , fRMsr sub , fRMsr ox , and fRMsr isopro crystals diffracted to 1.9, 2.3, 1.5, and 1.7 Å, respectively. The detailed statistics are summarized in Table 1.
Model Building and Structure Refinement-The crystal structures of fRMsr were solved by molecular replacement methods using CNS (28) and Molrep (31) programs. The coordinates of E. coli fRMsr (Protein Data Bank code 1VHM) (18,26) were used as the search model. Refinements were performed with several cycles of torsion-angle-simulated annealing, energy minimization, individual B factor refinement, and manual model rebuilding. The models were completed by iterative cycles of model building with Coot (32) and subsequently by refinement with CNS (28). The final models for fRMsr red , fRMsr sub , fRMsr ox , and fRMsr isopro yielded R factor and R free values of 21.6 and 25.6% for fRMsr red , 22.2 and 25.2% for fRMsr sub , 22.1 and 23.4% for fRMsr ox , and 22.0 and 24.2% for fRMsr isopro , respectively. Refinement data were validated by the PROCHECK program (33) and are provided in Table 1. All figures were created using CCP4mg (34).
Measurements of Msr Activities-For free Msr activity, NADPH oxidation was monitored as a decrease of A 340 at room temperature for 10 min in the reaction mixture. The reaction mixture (200 l) contained 50 mM sodium phosphate (pH 7.5), 50 mM NaCl, 0.2 mM NADPH, 10 g of E. coli Trx (Sigma), 14 g of human Trx reductase 1, 0.1 mM EDTA, 1 mM free Met-R-O or free Met-S-O, and 2 or 10 g of fRMsr enzyme. Enzyme activity was defined as nmol of oxidized NADPH/min using a molar extinction coefficient of 6220 M Ϫ1 cm Ϫ1 . K m and V max values were determined by non-linear regression using GraphPad Prism 5 software.
For peptide Msr activity, dabsylated methionine sulfoxide was used as the substrate in a DTT-dependent reaction. The reaction mixture (100 l), containing 50 mM sodium phosphate (pH 7.5), 50 mM NaCl, 20 mM DTT, 200 M dabsyl-Met-R-O or dabsyl-Met-S-O, and 1 g of fRMsr enzyme, was incubated at 37°C for 30 min. The reaction product, dabsyl-Met, was analyzed by high pressure liquid chromatography.
For inhibition assays of various alcohols on fRMsr activity, the reaction mixture (200 l) contained 50 mM sodium phosphate (pH 7.5), 50 mM NaCl, 0.2 mM NADPH, 10 g of E. coli Trx, 14 g of human Trx reductase 1, 0.1 mM EDTA, 1 mM free Met-R-O, 1% various alcohols, and 2 g of fRMsr enzyme. The AUGUST 6, 2010 • VOLUME 285 • NUMBER 32 reaction mixture was incubated at room temperature for 10 min, and the decrease of A 340 was monitored.

Catalytic Activities of Wild Type and Mutant
To determine the roles of the three conserved Cys residues (Cys 68 , Cys 78 , and Cys 102 ; supplemental Fig. S1) in catalysis, we mutated these residues to Ser, making single or double mutants (C68S, C78S, C102S, C68S/C78S, and C68S/C102S). We assayed the Trx-dependent activities of these mutant fRMsrs and compared them with the wild type. As shown in Table 2, the activity of C68S was 32% of wild type. Unexpectedly, the activity of C78S was 75% of wild type. This Cys residue was previously suggested to be the catalytic residue in E. coli and S. cerevisae fRMsrs (18,19). Interestingly, C102S had no catalytic activity. Consistent with this result, C68S/C102S had no catalytic activity either, whereas C68S/C78S retained 22% of enzyme activity.
We then analyzed kinetic parameters of C78S, C68S, and C68S/C78S as well as wild type ( Table 2). The V max value of C78S was slightly higher than that of wild type; the K m value was 2-fold higher than that of wild type. These data indicate that Cys 78 is non-essential for catalysis by fRMsr. The V max value was significantly reduced in C68S mutant, whereas the K m value was 4-fold higher, compared with those of wild type. The double C68S/C78S mutant exhibited more decreased V max (3-fold lower than wild type) and more increased K m (16-fold higher than wild type).
Thus, in contrast to the previously suggested model, Cys 102 is proposed to be the catalytic Cys, Cys 68 may serve as the resolving Cys that forms a disulfide bond with Cys 102 , and Cys 78 is a non-essential residue for catalytic function. The above enzymatic data are consistent with our recent findings from S. cerevisae fRMsr that Cys 125 (corresponding to Cys 102 in S. aureus fRMsr) functions as the catalytic residue, as determined by enzyme and in vivo growth complementation assays (35).
Crystal Structure of the Reduced Form of fRMsr-Previously known structures from both E. coli and S. cerevisae fRMsrs are oxidized forms with a disulfide bond between Cys 68 and Cys 102 (26,27). In addition, the E. coli fRMsr structure contains a complex with MES in the active site. Here, we resolved the structure of a reduced form of S. aureus fRMsr (fRMsr red ) (supplemental Fig. S2A). The crystal of fRMsr red comprises four dimers in the asymmetric unit. There are several hydrogen bond inter-  actions in the interface region of the dimer structure (Fig. 1A).
To assess on quantitative grounds the possibility that these hydrogen bond interactions may stabilize an fRMsr red dimer, the dimer interface was evaluated by using the program PISA (36). This widely used program estimates a dimeric state for fRMsr red (complexation significance score ϭ 1). In particular, this analysis shows that the buried area upon formation of the dimeric assembly is 932.7 Å 2 , which accounts for 11.8% of the total surface area for each molecule. It should be noted that S. cerevisae fRMsr is also a dimer in solution (27). The carboxamide groups of Asn 32 and Gln 63 from one subunit form hydrogen bonds with the backbones of Ala 67 and Phe 62 from the other subunit, respectively. In addition, the side chain of Gln 63 of one subunit interacts with the backbone of Gly 64 of the other subunit. The overall one-subunit structure of fRMsr red is composed of six antiparallel ␤-strands (␤1-␤6) and four ␣-helices (␣1-␣4) (supplemental Fig. S2A). The active site contained Trp 46 , Tyr 50 , Leu 59 , Cys 68 , Cys 78 , Cys 102 , Asp 103 , Ala 104 , Ser 106 , Glu 109 , Asp 125 , and Asp 127 in five antiparallel ␤-strands, two loops, and one ␣-helix, where Cys 78 is located (Fig. 2C and (Fig. 2C). These interactions involving water molecules in the active site may stabilize the conformation of reduced fRMsr.
Structure of fRMsr in Complex with the Substrate-Here, we have resolved the first structure of S. aureus fRMsr complexed with the substrate free Met-R-O (fRMsr sub ) using C68S fRMsr, which shows a Michaelis-like complex (supplemental Fig. S2B). The sulfoxide moiety of the substrate was clearly shown in the omit electron density map of the active site (Fig. 1B). This structure could lead us to understand the catalytic mechanism of fRMsr, the mode of binding to the substrate, and the roles of the active site residues during catalysis. The structure of fRMsr sub comprises a dimer with the substrate in each subunit of the asymmetric unit. The overall conformation of fRMsr sub , in which Ser replaces Cys 68 in the loop of the active site, is conserved with the reduced form of wild type fRMsr (supplemental Fig. S2). However, there are significant conformational changes around the active site, as discussed below.
The substrate Met-R-O is positioned by several hydrogen bonds and stacking interactions. The acidic side chains of  The backbone models for reduced (fRMsr red ), substrate-bound (fRMsr sub ), and oxidized (fRMsr ox ) forms are shown in green, light blue, and light yellow, respectively. B and C, comparison of active sites between fRMsr red (green) and fRMsr sub (light blue). The active site residues of fRMsr red and fRMsr sub are superimposed (B), and those of fRMsr red are independently shown (C). In C, hydrogen bond interactions among water molecules and active site residues are indicated by dotted lines. D, comparison of active sites between fRMsr sub (light blue) and fRMsr ox (light yellow). The active site residues of fRMsr sub and fRMsr ox are superimposed. E, comparison of active sites between fRMsr red (green) and fRMsr ox (light yellow). The active site residues of fRMsr red and fRMsr ox are superimposed. The disulfide bond between Cys 68 and Cys 102 in fRMsr ox is represented by a yellow stick, and substrate Met-R-O in fRMsr sub is shown by a light green stick. F, conformational changes of fRMsr red , fRMsr sub , and fRMsr ox . The active site is shown with electrostatic surface models. The surfaces are colored according to the electrostatic potentials from Ϫ21 kiloteslas/e (red) to ϩ21 kiloteslas/e (blue). The electrostatic surface potentials were calculated by using APBS (37).
Glu 109 , Asp 125 , and Asp 127 in the hydrophilic region form hydrogen bonds with nitrogen of the substrate (Fig. 1B). In addition, the residue Tyr 50 forms a hydrogen bond with the carboxylate group of Met-R-O. The sulfoxide of the substrate is located close to Cys 102 , pointing toward the sulfur atom of Cys 102 (6.7 Å). The thiol of Cys 78 points toward the carboxylate of the substrate although located closer to the sulfoxide of the substrate (4.3 Å). Our structural analysis, along with the above enzymatic data, suggests that Cys 102 is the catalytic residue of fRMsr. The hydrophobic region involving Ala 104 in the active site could accommodate the ⑀-methyl group of the substrate via van der Waals interactions, whereas the hydrophilic region could orient the substrate in the active site via hydrogen bonds with the nitrogen of the substrate. Also, this hydrophilic region may play a role in stabilizing the protonated oxygen atom of the sulfoxide moiety during catalysis. Thus, the hydrophobic pocket of the active site is shown to be essential for binding affinity to the substrate, whereas the hydrophilic region seems important for binding specificity.
Structure of the Oxidized Form of S. aureus fRMsr and a Comparison with Known fRMsr Structures-We also determined the structure of an oxidized form of S. aureus fRMsr (fRMsr ox ) containing a disulfide bond between Cys 68 and Cys 102 (supplemental Fig. S2C). The structure of fRMsr ox comprises one subunit in the asymmetric unit. However, fRMsr ox is found to form a dimer with a crystallographic 2-fold symmetry-related molecule in the unit cell. In fact, the fRMsr red and fRMsr sub structures were grown in the p2 1 monoclinic space group, whereas the fRMsr ox crystal grew in the p6 1 22 hexagonal space group.
We compared the S. aureus fRMsr ox with the structures of E. coli and S. cerevisiae fRMsrs previously reported, which are also oxidized forms with a disulfide bond between the above Cys residues. The oxidized E. coli fRMsr contains MES in the active site. S. aureus fRMsr shows 53% amino acid sequence identity with E. coli and S. cerevisiae fRMsrs, respectively (supplemental Fig. S1). The backbone structure of S. aureus fRMsr ox could be superimposed on the E. coli and S. cerevisiae fRMsrs with r.m.s. deviations of 1.6 and 5.4 Å, respectively, as determined by CNS (Crystallography and NMR System) (28) for 154 C␣ atoms of the overall structures (supplemental Fig. S3A). Interestingly, there were significant differences in a loop region including the catalytic Cys 102 (residues 97-106) between S. aureus fRMsr ox and E. coli fRMsr (supplemental Fig. S3B). Particularly, positions of His 99 , Ala 101 , and Asp 103 move away from the corresponding residues of E. coli fRMsr to a distance of 3.3, 4.8, and 3.6 Å, respectively. Also, this loop region was significantly different from that of S. cerevisiae fRMsr (supplemental Fig. S3C). The positions of Ala 101 and Asp 103 move away from the corresponding residues of S. cerevisiae fRMsr to a distance of 4.2 and 6.1 Å, respectively. Thus, the structural comparison revealed that the catalytic Cyscontaining loop region is quite flexible in fRMsr proteins.
Comparison and Conformational Changes of Reduced, Substrate-bound, and Oxidized Forms of fRMsr-We compared the reduced (fRMsr red ), substrate-bound (fRMsr sub ), and oxidized (fRMsr ox ) structures of S. aureus fRMsr (Fig. 2), which are representative of the catalytic steps of the fRMsr reaction.
The backbone structure of fRMsr sub could be superimposed on the fRMsr red , with an r.m.s. deviation of 1.4 Å (Fig. 2A). There were significant conformational changes particularly in the loop consisting of residues 97-106 (Fig. 2B). Cys 102 and Asp 103 of fRMsr sub are the most displaced residues in the loop, shifted by 4.7 and 10.9 Å, respectively. The positions of Ile 100 , His 99 , and Lys 97 lie at 2.4, 5.1, and 2.2 Å, respectively, from the corresponding residues of fRMsr red . However, the position of Cys 78 in fRMsr sub and fRMsr red remains relatively unchanged. The Glu 109 residue in fRMsr sub resides at a distance of 3.6 Å from Asp 127 , whereas in fRMsr red it resides at a distance of 4.7 Å. Cys 68 and Cys 102 residues in fRMsr sub reside at a distance of 9.7 and 11.7 Å, respectively, from Cys 78 , whereas in fRMsr red they reside at a distance of 10.1 and 10.2 Å, respectively. Water molecules in the active site of fRMsr red interact with Glu 109 , Asp 125 , Asp 127 , and Tyr 50 residues that form hydrogen bonds with nitrogen and the carboxylate group of the substrate (Fig.  2C). When comparing the structure of fRMsr sub with fRMsr red , the substrate Met-R-O in the active site replaces the water molecules occupied in the fRMsr red (Fig. 2, B and C).
We next compared the structure of fRMsr sub with fRMsr ox . The backbone structure of fRMsr sub could be superimposed on the fRMsr ox , with an r.m.s. deviation of 1.4 Å ( Fig. 2A). Significant conformational changes are observed in the loop of residues 97-106 between these two structures (Fig. 2D). Cys 102 and Asp 103 residues are shifted by 6.7 and 7.8 Å, respectively, between fRMsr sub and fRMsr ox . Specifically, in the substrate complex form, the loop moves into the active site compared with the oxidized form, resulting in positioning the thiol group of the catalytic Cys 102 toward the entrance of the active site. The positions of Ile 100 , His 99 , and Gly 98 in fRMsr ox lie at 6.6, 7.7, and 3.4 Å, respectively, from the corresponding residues of fRMsr sub . However, the position of Cys 78 in fRMsr sub and fRMsr red remains relatively unchanged.
We finally compared fRMsr red structure with fRMsr ox structure. The backbone structure of fRMsr red could be superimposed on the fRMsr ox with an r.m.s. deviation of 1.5 Å (Fig. 2A). Although the overall structures were well superimposed, there were significant differences in the loop of residues 94 -106 (Fig.  2E). The loop in fRMsr red moves into the active site, which results in the positioning of the His 99 , Cys 102 , Asp 103 , and Ala 104 residues toward the entrance of active site. Large movements occur in the catalytic residue Cys 102 and its neighboring residues Asp 103 and Ala 104 . The Cys 102 residues in the oxidized and reduced forms of fRMsr reside at a distance of 4.7 Å from each other. Asp 103 and Ala 104 in the oxidized and reduced forms reside at a distance of 8.7 and 6.5 Å, respectively, from each other. The side chains of Glu 109 and Asp 127 in the active site reside farther from each other in fRMsr red (4.9 Å) than in fRMsr ox (3.7 Å). Moreover, the distance between Cys 78 and Asp 127 is changed from 5.8 Å in fRMsr red to 8.3 Å in fRMsr ox . In addition, the fRMsr red structure shows movement of the side chain of Cys 68 toward the entrance of the active site. Together, the movements of the active site residues (particularly Cys 102 ) determine the conformations of fRMsr red and fRMsr ox , leading to an open conformation in fRMsr red and a closed conformation in fRMsr ox (Fig. 2F). AUGUST 6, 2010 • VOLUME 285 • NUMBER 32

Structure and Catalytic Mechanism of fRMsr
Catalytic Mechanism-Our crystal structures of fRMsr red , fRMsr sub , and fRMsr ox help in understanding the mode of binding of the substrate Met-R-O to the active site of fRMsr, the roles of the active site residues, and the conformational changes of fRMsr during catalysis. Significant conformational changes of the active site, particularly in the loop including the catalytic Cys, occur in each catalytic step. The reduced form has an open conformation to allow access to the substrate, the substratebound form takes a closed conformation after accommodation of Met-R-O, and the oxidized form is turned to a more closed conformation after catalysis (Fig. 2F).
Among the three conserved Cys residues, Cys 102 was the most mobile, whereas Cys 78 , the previously suggested catalytic residue from E. coli and S. cerevisiae fRMsrs (18,19), was the most immobile. Our enzymatic studies concluded that Cys 102 is the catalytic residue. Cys 68 is suggested to be the resolving Cys by structural and kinetic analyses. Cys 78 had no catalytic function, but this residue may play a role in substrate binding, as judged by the kinetic data (i.e. an increase in K m value in the C78S mutant). Here, we propose that the catalytic mechanism of fRMsr consists of three steps. 1) Cys 102 attacks the sulfoxide moiety of Met-R-O and is then oxidized to Cys sulfenic acid. 2) Cys 68 interacts with the sulfenic acid intermediate to form a disulfide bond. 3) Finally, the Cys 102 -Cys 68 disulfide bond is reduced by a reductant (typically by Trx), and the fRMsr enzyme activity is regenerated (Fig. 3).
It should be noted that, in contrast to type I fRMsrs, type II enzymes contain only the conserved Cys 78 and Cys 102 . They lack Cys 68 . Because our studies revealed no direct function for Cys 78 in the catalysis of type I fRMsr, it is questionable whether this residue plays any role in the catalysis of type II fRMsr. It is possible that this Cys 78 would function as a resolving Cys, replacing the role of Cys 68 in type I enzymes. Thus, biochemical and structural studies of type II fRMsr would be interesting.
Implications of the Mechanism of Action of fRMsr Using a Competitive Inhibitor-We determined another complex structure (fRMsr isopro ) that contains isopropyl alcohol in the active site pocket of fRMsr. This crystal was obtained from a crystallization solution consisting of 2 M ammonium sulfate and 10% (v/v) isopropyl alcohol and had one subunit of protein in the asymmetric unit like fRMsr ox . The fRMsr isopro structure also contains a disulfide bond formed by Cys 68 and Cys 102 . Interestingly, the binding pattern of isopropyl alcohol is expected to define the location of the substrate binding site (Fig. 4). The hydroxyl group of isopropyl alcohol interacts by hydrogen-bonding with Glu 109 , Asp 125 , and Asp 127 , respectively. This is similar to the hydrogen bond interactions of these residues with the nitrogen of the substrate in fRMsr sub . This structural analysis suggests that isopropyl alcohol could act as a competitive inhibitor of fRMsr enzyme. To test this hypothesis, we assayed the enzyme activities in the presence of 1% ethanol, n-propyl alcohol, or isopropyl alcohol (Table 3). Relative activities of fRMsr in the presence of ethanol and n-propyl alcohol were 91%. However, in the presence of isopropyl alcohol, the enzyme activity significantly decreased to 69%. We further determined kinetic parameters in the presence of 1% isopropyl alcohol. The K m value was 100 Ϯ 30 M, which is 2-fold higher than that in the absence of isopropyl alcohol. The V max value was 420 Ϯ 30 nmol/min/mg of protein, similar to that without isopropyl alcohol. These results indicated that isopropyl alcohol can competitively inhibit fRMsr activity. Moreover, a branched methyl group in isopropyl alcohol may play a role in this inhibitory effect.
We further tested the inhibitory effect with 1% n-butyl alcohol and isobutyl alcohol (Table 3). In the presence of n-butyl alcohol, the relative activity was 87%, similar to that with ethanol or n-propyl alcohol. However, the enzyme activity was significantly inhibited by 50% in the presence of isobutyl alcohol. Together, our results indicate that a branched methyl group in alcohols seems important for competitive inhibition of fRMsr enzyme activity. The branched methyl group of alcohols may be crucial for binding to the active site in order to competitively inhibit the fRMsr activity, suggesting that the methyl group of the substrate may be important for binding affinity to the enzyme shown in the structure of fRMsr sub .
In summary, we have determined the crystal structures of reduced, substrate-bound, oxidized, and inhibitor-bound fRMsrs at atomic resolution levels. Our structural and biochemical studies suggest the catalytic mechanism of fRMsr, where Cys 102 acts as the catalytic residue and Cys 68 acts as the resolving Cys. Our structures show the mode of binding of the substrate free Met-R-O, the roles of active site residues in catalysis, and the conformational changes of the active site during catalysis, particularly by the loop containing the catalytic Cys 102 . In addition, our studies with a competitive inhibitor, isopropyl alcohol, predict the mechanism of action of fRMsr, where the methyl group in the substrate or a branched methyl group in alcohols seems important for interaction with the enzyme.