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Originally published In Press as doi:10.1074/jbc.M513754200 on February 16, 2006

J. Biol. Chem., Vol. 281, Issue 15, 10533-10539, April 14, 2006
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Identification of the Amino Acid Residues Essential for Proteolytic Activity in an Archaeal Signal Peptide Peptidase*

Rie Matsumi, Haruyuki Atomi, and Tadayuki Imanaka1

From the Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan

Received for publication, December 27, 2005 , and in revised form, February 10, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Signal peptide peptidases (SPPs) are enzymes involved in the initial degradation of signal peptides after they are released from the precursor proteins by signal peptidases. In contrast to the eukaryotic enzymes that are aspartate peptidases, the catalytic mechanisms of prokaryotic SPPs had not been known. In this study on the SPP from the hyperthermophilic archaeon Thermococcus kodakaraensis (SppATk), we have identified amino acid residues that are essential for the peptidase activity of the enzyme. {Delta}N54SppATk, a truncated protein without the N-terminal 54 residues and putative transmembrane domain, exhibits high peptidase activity, and was used as the wild-type protein. Sixteen residues, highly conserved among archaeal SPP homologue sequences, were selected and replaced by alanine residues. The mutations S162A and K214A were found to abolish peptidase activity of the protein, whereas all other mutant proteins displayed activity to various extents. The results indicated the function of Ser162 as the nucleophilic serine and that of Lys214 as the general base, comprising a Ser/Lys catalytic dyad in SppATk. Kinetic analyses indicated that Ser184, His191 Lys209, Asp215, and Arg221 supported peptidase activity. Intriguingly, a large number of mutations led to an increase in activity levels of the enzyme. In particular, mutations in Ser128 and Tyr165 not only increased activity levels but also broadened the substrate specificity of SppATk, suggesting that these residues may be present to prevent the enzyme from cleaving unintended peptide/protein substrates in the cell. A detailed alignment of prokaryotic SPP sequences strongly suggested that the majority of archaeal enzymes, along with the bacterial enzyme from Bacillus subtilis, adopt the same catalytic mechanism for peptide hydrolysis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Secretion proteins and membrane proteins in many cases harbor signal sequences at their extreme N termini that are cleaved during translocation by signal peptidases (1). The released signal peptides are subsequently cleaved into smaller fragments by signal peptide peptidases (SPPs)2 (2). It had long been presumed that the signal peptides, after their removal from the precursor protein, have no active function in the cell and are simply degraded to free amino acids. However, it is now known that in human cells, peptide fragments generated after cleavage by SPP exhibit vital regulatory functions in immune surveillance (3, 4). The function of SPP has also been reported to be necessary for the proper development of Drosophila larvae (5).

In bacteria, SPP was first identified in Escherichia coli. A previously identified cytoplasmic membrane protein, protease IV (6), was found to exhibit SPP activity toward the signal peptide of the outer membrane lipoprotein (2). The gene was cloned (sppAEc) (7), and gene disruption studies strongly implied the involvement of SppAEc in signal peptide degradation (8). Other cytoplasmic proteases including oligopeptidase A have also been found to participate in this degradation pathway (9). It is now presumed that in E. coli, SppA initiates the degradation by introducing endoproteolytic cuts into the signal peptide, whereas the other cytoplasmic proteases are responsible for complete degradation of the smaller fragments into free amino acids (10). SppA has also been identified and genetically characterized in the Gram-positive Bacillus subtilis. The enzyme, along with a cytoplasmic peptidase TepA, has been found to play an important role in signal peptide degradation in this organism (11).

In the Archaea, much remains to be understood on the mechanisms of protein secretion (12-14) and the fate of the signal peptide after its release from the precursor protein. In terms of signal peptidases, the type I signal peptidase from Methanococcus voltae has been characterized, and a catalytic triad comprised from Ser52, His122, and Asp148 has been determined to be critical for its peptidase activity (15, 16). FlaK, an aspartic protease essential for preflagellin signal cleavage has also been studied from this organism (17). In the Crenarchaeota, the homologue of bacterial type IV prepilin peptidases from Sulfolobus solfataricus (PibD) has been characterized, and residues on the substrate that are important for recognition by PibD have been examined (18).

As for SPPs, we have recently carried out the first examination of an archaeal SPP (SppATk) (19) from the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 (20). SppATk (334 residues) was much smaller in size compared with its bacterial counterpart SppAEc (618 residues). A single, putative membrane-spanning domain was present in the N-terminal region of the protein. We found that {Delta}N54SppATk, a truncated protein without the N-terminal 54 residues, was a soluble protein exhibiting peptidase activity and stable against autoproteolysis. The substrate specificity of {Delta}N54SppATk examined with a FRETS peptide library was consistent with its presumed role as an SPP in T. kodakaraensis.

From the primary structures, archaeal and bacterial homologues of SppATk and SppAEc are all members of the S49 family of peptidases included in the Clan SK (MEROPS, the peptidase data base, merops.sanger.ac.uk/) (21). Although the eukaryotic SPPs have been determined to be aspartic proteases (22, 23), the effects of various inhibitors on SppATk and SppAEc strongly suggest that the enzymes are serine proteases (6, 19). Although sequence comparisons among SppA homologues reveal the presence of several conserved serine residues, experimental evidence identifying the nucleophilic serine has not yet been obtained. Moreover, even with the sequence comparisons, it is still difficult to estimate what other residues might be involved in the catalytic mechanism of SPPs. In particular, His and Asp/Glu residues that comprise the well known catalytic triad of serine proteases are not clearly conserved among the bacterial and archaeal SppA sequences.

To gain insight on the residues involved in the catalytic mechanism of prokaryotic SPPs, here we have performed a detailed site-directed mutagenesis study on {Delta}N54SppATk. Through the analyses of various mutant proteins, we have been able to determine multiple residues that are essential or important for the activity of this protein. Our results strongly suggest that {Delta}N54SppATk and other SppA homologues from the Archaea utilize a Ser/Lys dyad mechanism in peptide cleavage.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains, Media, and Plasmids—E. coli BL21-CodonPlus(DE3)-RIL (Stratagene, La Jolla, CA) was used as the host strain for gene expression with the plasmid pET21a(+) (Novagen, Madison, WI). The plasmids were amplified with E. coli JM109 (Toyobo, Osaka, Japan). E. coli strains were cultivated in LB medium (10 g/liter of tryptone, 5 g/liter of yeast extract, and 10 g/liter of NaCl) with 100 µgml-1 ampicillin at 37 °C.

DNA Manipulation, Sequence Analysis, and Site-directed Mutagenesis—Plasmid DNA was isolated with the plasmid mini kit from Qiagen. DNA sequencing was performed using a BigDye terminator cycle sequencing kit v.3.1 and a model 3100 capillary DNA sequencer (Applied Biosystems, Foster City, CA). Sequence comparisons and alignments were performed with the ClustalW program provided by the DNA Data Bank of Japan. Site-directed mutagenesis was carried out using a QuikChange XL site-directed kit (Stratagene). The template used was an expression plasmid previously constructed for wild-type {Delta}N54SppATk, a protein with an N-terminal truncation of 54 amino acid residues. An artificial Met residue was inserted directly before Cys55 (19). The primers used to incorporate each mutation are shown in Table 1. After sequence confirmation, the plasmids were introduced into E. coli BL21-CodonPlus(DE3)-RIL cells.


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  		TABLE 1
Primers used in this study

 
Expression and Purification of Wild-type and Mutant {Delta}N54SppATk Proteins—The recombinant E. coli cells were grown in LB medium, and gene expression was induced with 0.1 mM isopropyl-beta-D-thiogalactopyranoside at the mid- to late exponential growth phase. After 6 h, the cells were collected, washed with 50 mM Tris-HCl (pH 8.0), and resuspended in the same buffer. The cells were sonicated on ice, and the supernatant after centrifugation (20,000 x g, 30 min at 4 °C) was applied to heat treatment at 85 °C for 15 min, immediately cooled on ice, and then centrifuged (20,000 x g, 30 min at 4 °C). The soluble protein sample was brought to 35% saturation with ammonium sulfate, and the precipitate was dissolved in 50 mM Tris-HCl (pH 8.0). This was applied to anion exchange chromatography (ResourceQ; Amersham Biosciences) equilibrated with 50 mM Tris-HCl (pH 8), 0.2 M NaCl and eluted with a linear gradient (0.2-1.0 M) of NaCl. After desalting with a HiPrep26/10 column (Amersham Biosciences), the sample was applied to gel filtration chromatography (Superdex 200 HR 10/30; Amersham Biosciences) equilibrated with 50 mM Tris-HCl (pH 8.0), 0.15 M NaCl, and the obtained fractions were used for enzyme analysis. The protein concentration was determined with a protein assay kit (Bio-Rad) using bovine serum albumin as a standard.

Enzyme Activity Measurements—Standard activity measurements were performed at 60 °C in 1 ml of 50 mM CHES (pH 10.0) with 0.1 µg of purified protein and 200 µM Ala-Ala-Phe-MCA available from Peptide Institute (Osaka, Japan). Release of 7-amino-4-methyl-coumarin was monitored consecutively with a fluorescence spectrophotometer capable of maintaining the cuvette at desired temperatures between 30 and 100 °C. {lambda}ex and {lambda}em were 380 and 460 nm, respectively. The final concentration of Me2SO used to dissolve the substrate was constant at 3% of the reaction mixture. Kinetic parameters were calculated with IGOR Pro version 5.0 (WaveMetrics, Lake Oswego, OR).

Circular Dichroism Spectroscopy of Wild-type and Mutant Enzymes—Each protein sample was prepared in 25 mM Tris-HCl (pH 8.0), 75 mM NaCl at a protein concentration of ~0.1 mg ml-1. A J-820 spectropolarimeter (Jasco, Tokyo, Japan) was used to measure ellipticity as a function of wavelength from 250 to 200 nm in 0.2-nm increments using a 0.1-cm cylindrical quartz cuvette. The samples were scanned one hundred times and averaged. The mean molar ellipticity [{theta}] (deg cm2 dmol-1) was calculated from the equation [{theta}] = {theta}/10 nCl, where {theta} is the measured ellipticity in millidegrees, C is the molar concentration of enzyme subunits, l is the path length in centimeters, and n is the number of residues/subunit.

Examination of the Substrate Preference of Wild-type and Mutant Enzymes—The substrate preferences of the wild-type, S128A, and Y165A proteins were examined with a FRETS peptide library (25Xaa series, Peptide Institute) (19, 24). These peptide substrates harbor a highly fluorescent 2-(N-methylamino)benzoyl group linked to the side chain of the N-terminal D-2,3-diamino propionic acid residue (D-A2pr), along with a 2,4-dinitrophenyl group (quencher) linked to the {epsilon}-amino group of a Lys residue. In between the D-2,3-diamino propionic acid residue and the Lys residue lies the peptide Gly-Zaa-Yaa-Xaa-Ala-Phe-Pro, where Zaa is a mixture of Phe, Ala, Val, Glu, or Arg; Yaa is a mixture of Pro, Tyr, Lys, Ile, or Asp; and the Xaa residue is a defined single amino acid of choice. {lambda}ex and {lambda}em were 340 and 440 nm, respectively. To examine the preference for residues at the P-1 position, 1 µg of purified enzyme was added to the reaction mixture with a final volume of 1 ml containing 30 µM substrate in 50 mM CHES (pH 10.0). The final concentration of Me2SO used to dissolve the substrate was constant at 3% of the reaction mixture.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Highly Conserved Amino Acid Residues among the Signal Peptide Peptidases from Archaea and Bacteria—To determine which residues should be selected for site-directed mutagenesis and subsequent biochemical analyses, we aligned all SppA homologues found in the archaeal genomes, along with the biochemically and/or genetically characterized bacterial SppA from E. coli and B. subtilis. Among the 21 archaeal genomes that have been sequenced, we could identify 19 SppA homologues in 16 organisms (see legend of Fig. 1). Although a number of other open reading frames have been annotated as putative SPPs, they displayed clearly lower degrees of similarity with SppATk and SppAEc and were therefore not selected for further examination. The sequence comparison indicated that SppA homologues were not present in Aeropyrum pernix, S. solfataricus, and Sulfolobus tokodaii from the Crenarchaeota and Methanopyrus kandlerii and Archaeoglobus fulgidus from the Euryarchaeota. A region corresponding to residues between Met115 and Lys291 in SppATk was fairly conserved among all sequences and presumably includes the catalytic core regions of these enzymes. An alignment of the sequences spanning this region, along with the mutations introduced in this study, is shown in Fig. 1.


Figure 1
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  		FIGURE 1.
An amino acid sequence alignment of the core regions of archaeal and bacterial homologues of SppATk and SppAEc. Nineteen archaeal SppA sequences were aligned along with the sequences of SppA from E. coli and B. subtilis. The conserved residues selected for site-directed mutagenesis are indicated with arrowheads and numbered. Residues identical with those of SppATk are indicated in red. The abbreviations of the proteins (italicized) and accession numbers of all sequences used for the alignment are as follows:B. subtilis SppA (CAB14931 [GenBank] , Bsu), E. coli (BAA15557 [GenBank] , Eco), H. marismortui (I, AAV45638 [GenBank] , Hm1; II, AAV46904 [GenBank] , Hm2; III, AAV47811 [GenBank] , Hm3), Halobacterium sp. NRC-1 (AAG19125 [GenBank] , Hba), M. jannaschii (AAB98642 [GenBank] , Mja), Methanococcus maripaludis (CAF30625 [GenBank] , Mmr), Methanosarcina acetivorans (AAM07395 [GenBank] , Mac), Methanosarcina mazei (AAM30562 [GenBank] , Mma), Methanothermobacter thermautotrophicus (AAB85306 [GenBank] , Mth), N. equitans (AAR39164 [GenBank] , Neq), Picrophilus torridus (AAT42796 [GenBank] , Pto), Pyrobaculum aerophilum (I, AAL65089 [GenBank] , Pa1; II, AAL64441 [GenBank] , Pa2), Pyrococcus abyssi (CAB49512 [GenBank] , Pab), Pyrococcus furiosus (AAL81707 [GenBank] , Pfu), P. horikoshii (BAA30681 [GenBank] , Pho), T. kodakaraensis (BAD85353 [GenBank] , Tko), Thermoplasma acidophilum (CAC11222 [GenBank] , Tac), and Thermoplasma volcanium (BAB59171 [GenBank] , Tvo). The division of the sequences into two groups is described under "Discussion."

 
Although the eukaryotic SppA has been demonstrated to be an aspartate peptidase (22), inhibitor studies on the bacterial SppAEc and the archaeal SppATk have indicated that both of these enzymes are serine peptidases (6, 19). We therefore initially focused on the presence of conserved serine residues. Ser128, Ser162, and Ser184 of SppATk were completely conserved in all sequences, with the only exceptions being a replacement of the first Ser to Thr in one of the three homologues from Haloarcula marismortui and a replacement of the third Ser to His in another homologue from this strain. In terms of the His residue in the well known Ser/His/Asp catalytic triad, only three His residues were found in this region of SppATk and were not highly conserved among the SppA sequences. His140 and His213 were not conserved even in the closely related Pyrococcus spp., whereas His191, although shared by SppATk and the three Pyrococcus enzymes, was not found in the other SppA sequences. As an alternative, we found that the basic residue Arg250 was highly conserved among the SppA proteins. As for the Asp residue, Asp277 was the only acidic residue that was present without exception in all sequences (Fig. 1).

Production and Purification of Mutant SppATk Proteins via Site-directed Mutagenesis—Based on the sequence alignment described above, we first constructed the following six mutants; S128A, S162A, S184A, H191A, R250A, and D277A. The primers used to incorporate the mutations are shown in Table 1. The mutations were incorporated into the expression vector for {Delta}N54SppATk, a truncated protein without the N-terminal 54 residues of SppATk. Sequence analysis confirmed that only the intended mutations were introduced into the genes. Recombinant mutant proteins were produced in E. coli BL21-CodonPlus(DE3)-RIL cells and purified from the cell-free extracts by heat treatment at 85 °C for 15 min, followed by ammonium sulfate fractionation, anion exchange chromatography, and gel filtration chromatography. Although we were able to obtain each mutant protein in a soluble form, the amount of protein produced in the E. coli cells varied with each mutant. We previously reported that the recombinant {Delta}N54SppATk was obtained in an octameric or hexadecameric form (19). We also observed various quaternary structures in the mutant proteins, but only the fraction corresponding to the octameric form of the protein was used for further analysis. The apparent homogeneity of each protein after the purification procedure was examined by SDS-PAGE (Fig. 2).


Figure 2
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  		FIGURE 2.
SDS-PAGE of the wild-type and mutant proteins. The wild-type (WT) {Delta}N54SppATk and 16 mutant proteins with single amino acid replacements were subjected to SDS-PAGE after the purification procedures described under "Experimental Procedures." Mobility of molecular mass markers is indicated on the left side of each gel.

 


Figure 3
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  		FIGURE 3.
Relative activity level of each mutant protein toward Ala-Ala-Phe-MCA. Substrate concentration was fixed at 200 µM, and the reaction temperature was 60 °C. The activity level of the wild-type (WT) {Delta}N54SppATk was designated as 100%. The error bars indicate standard deviation.

 
Peptidase Activity of the Wild-type and Mutant {Delta}N54SppATk Proteins—We have previously reported that the synthetic peptide Ala-Ala-Phe-MCA is a good substrate for the wild-type {Delta}N54SppATk (19). The activity levels of each mutant protein were measured at 60 °C in the presence of a fixed concentration of this substrate (200 µM) and compared with that of the wild-type enzyme (Fig. 3). We found that the mutation of Ser162 had the most detrimental effect. No activity could be observed with the standard assay procedures. We could estimate that the activity of the protein was only 0.004% of that of the wild-type enzyme by using a 100-fold higher protein concentration. The S184A and H191A mutations also brought about a decrease in enzyme activity, but significant levels of activity, 24 and 45% of that of the wild-type enzyme, respectively, were still observed in each mutant protein. Interestingly, the S128A, R250A, and D277A mutations did not have any negative effect but rather led to an unexpected increase in peptidase activity. It has been shown that mutations in the catalytic residues of serine proteases with a catalytic triad result in 104-106-fold reduction in activity (25). Our results clearly indicate that Ser162 is essential for the peptidase activity of SppATk and is most likely the nucleophilic serine of the enzyme. The results also reveal that Arg250 and Asp277, two highly conserved residues in both archaeal and bacterial SPPs, have little, if any, role in the peptide hydrolyzing mechanism of SppATk.

Mutations to Identify the General Base Residue of SppATk—The absence of conserved histidine residues among SppA proteins and the fact that His191 was not essential for the catalytic activity suggested that SppATk was not dependent on the well known Ser/His/Asp catalytic triad. We therefore searched for other basic and acidic residues as targets for site-directed mutagenesis. Intriguingly, there were no other charged residues that were completely conserved in all of the sequences. We therefore chose those that were the most highly conserved among the sequences, Lys150, Lys209, Lys214, Asp215, Arg221, Glu226, and Glu227. Each residue was replaced by Ala with appropriate primers (Table 1). Expression and purification were carried out as described for the initial mutant proteins, and the apparent homogeneity of each mutant protein is displayed in Fig. 2.

The K150A and E226A mutations did not have a negative effect on the activity of the enzyme. All other residue replacements led to mutant proteins with lower levels of activity than that of the wild-type enzyme. Moderate effects were observed in K209A (59% activity retained), E227A (35%), and D215A (20%), whereas the effect of the R221A (4%) was much more significant. The K214A mutation had the most dramatic effect, and as in the case of the S162A mutation, activity could not be detected under standard procedures. By applying increased protein concentration, activity of K214A was estimated at 0.01% of that of the wild-type protein. The results indicate that Lys214 is essential for activity of SppATk and that the protein most likely utilizes a Ser-Lys catalytic dyad for peptide cleavage. The results also indicate that Arg221, although not essential, plays an important role in the peptidase activity of SppATk.


Figure 4
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  		FIGURE 4.
Substrate concentration-reaction velocity plots for wild-type (WT) {Delta}N54SppATk and its mutants K150A, Y165A, and H191A. The measurements were performed at 60 °C. The data for each protein were fitted with the equation v = Vmax[S]/(Ks1 +[S] + 1/Ks2 [S]2).

 
Effects of Mutations on Other Highly Conserved Residues—We next focused on conserved, noncharged residues. Several glycine residues were highly conserved among the homologues, and in particular, a consecutive Gly-Gly sequence was conserved in all SppA proteins. In some lipases and esterases, a consecutive glycine sequence provides the main chain amino group(s) that stabilize the oxyanion intermediate that is formed after the nucleophilic attack on the carbonyl group by serine (26). We therefore constructed the mutant proteins G130A and G131A. We also selected a Tyr residue in the near vicinity of the nucleophilic Ser162 that is completely conserved among Spp proteins and introduced a Y165A mutation. As a result, we observed a moderately positive effect with the G130A mutation and a surprisingly high increase in activity in the Y165A mutant protein. The G131A mutation had a drastic effect on activity, and the protein exhibited only 2% of the activity levels of the wild-type enzyme, indicating an important role for this glycine residue.

Circular Dichroism Spectra of Wild-type and Mutant {Delta}N54SppATk Proteins—To examine whether the single-residue replacements had unintended broad effects on the protein structure, we analyzed the CD spectrum of each mutant protein. In all mutant proteins, including the two that hardly exhibited activity (S162A and K214A), the CD spectra were indistinguishable from that of the wild-type protein (data not shown). We would like to note that in the case of G130A and G131A, we observed a slight increase in ellipticity between 225 and 230 nm, raising the possibility of protein-protein interaction. Overall, the results indicate that the single-residue replacements introduced did not lead to significant changes in the secondary structures of the proteins. This allows us to interpret the changes in activity as direct consequences brought about by residue exchange.

Kinetic Analysis of the Mutant Proteins—We next performed kinetic analyses for all mutant proteins that exhibited sufficient levels of activity. Most mutant proteins were analyzed by standard procedures with various concentrations of Ala-Ala-Phe-MCA. The S162A and K214A mutant proteins could not be examined because of their extremely low levels of activity. Analyses of the G131A and R221A proteins were carried out with 20- and 10-fold higher amounts of enzyme, respectively.

We have previously reported that the cleavage reaction of Ala-Ala-Phe-MCA by the wild-type {Delta}N54SppATk followed Michaelis-Menten kinetics (19). However, in the process of analyzing the activity levels of mutant proteins at high substrate concentrations, we observed a decrease in activity levels. Further examination of all active proteins, including the wild-type {Delta}N54SppATk, confirmed the occurrence of substrate inhibition. By considering several equations, we found that our data fit very well (R2 > 99.4) to one of the typical substrate inhibition models, expressed as v = Vmax[S]/(Ks1 + [S] + 1/Ks2 [S]2), where v is reaction velocity, Vmax is maximum velocity, [S] is substrate concentration, Ks1 is the dissociation constant between enzyme and the first substrate, and Ks2 is the dissociation constant between the enzyme-substrate complex and the second, inhibitory substrate. Representative [S]-v plots with the respective curves are shown for the wild-type and K150A, Y165A, and H191A mutant proteins in Fig. 4. The kinetic parameters of each protein are indicated in Table 2.


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  		TABLE 2
Kinetic parameters for wild-type and mutant proteins

 
When compared with the wild-type protein, a number of mutant proteins exhibited similar Ks1 values, indicating that these residue replacements did not greatly alter the affinity of the enzyme toward the substrate Ala-Ala-Phe-MCA. Significant increases in the Ks1 values were observed for S128A, G130A, G131A, D215A, R221A, and E227A. Because these mutations, other than G130A and G131A, result in a decrease in the size of the side chain, the increase in Ks1 values may be due to the abolishment of favorable interactions between the binding pocket and substrate. The increase may also be due to the hydrophobic property of the Ala residue, which may lead to a repulsive effect on a hydrophilic region of the substrate. As for G131A, reaction velocity continued to increase with increases in substrate concentration up to at least 3,000 µM, and therefore reliable curve fitting could not be performed. Fitting the equation

with the limited data indicated that the Ks1 value was higher than 106 µM. The G130A mutant also displayed Ks1 values >104 µM, and reliable values for the kinetic parameters could not be obtained. In these two proteins, the additional methyl group may directly hinder the binding between enzyme and substrate. On the other hand, the glycine residues may be important in maintaining the optimized structure of the protein, because consecutive glycine residues would allow dramatic bends in the main chain backbone. As mentioned above, we did observe subtle changes in the CD spectra of these two glycine mutant proteins. On the SDS-PAGE gels, we also noticed a slight difference in the mobility of G130A along with the appearance of a high molecular weight band in the case of G131A, both of which are most likely due to incomplete denaturation or dissociation of the oligomeric protein (Fig. 2). Although the CD spectra rule out drastic changes in protein conformation, mutations of these two residues may result in subtle changes in the enzyme conformation.

A rather unexpected result was the relatively large number of mutations that led to increases in the Vmax or kcat/Ks1 values compared with the wild-type enzyme. Significant increases in the Vmax value were observed in S128A (690%), K150A (148%), Y165A (232%), E226A (135%), R250A (146%), and D277A (159%). Even in the kcat/Ks1 values, which should better represent the functional capacity of the enzyme in the cell, notable increases were observed in S128A (155%), K150A (163%), Y165A (222%), E226A (135%), and R250A (169%). The fact that these conserved residues seem to suppress the activity levels of SppATk may be important for the proper function of the enzyme in vivo (see "Discussion").


Figure 5
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  		FIGURE 5.
Peptidase activity of wild-type {Delta}N54SppATk and its mutants S128A and Y165A toward various substrates from a FRETS peptide library. Amino acid residues at the Xaa position are indicated with single-letter abbreviations. Each substrate was examined at a concentration of 30 µM. The activity of the wild-type (WT) protein toward the Xaa = Gly substrate was designated as 100%.

 
Examination of the Activity of S128A and Y165A toward Various Substrates—Two mutant enzymes, S128A and Y165A, which displayed particularly large increases in both Vmax and kcat/Ks1 values, were selected for examination of their activities against a FRETS peptide library as described under "Experimental Procedures." We have previously examined the activity of the wild-type enzyme against these substrates and observed higher cleavage rates toward peptides with small amino acid residues at the Xaa position, corresponding to the P-1 position. Activity toward substrates with charged residues or aromatic residues at the Xaa position was low (19). We found that the wild-type enzyme exhibited the same tendencies in this study also (Fig. 5). When we performed the same activity measurements with the S128A and Y165A mutant proteins, we observed that activity levels toward substrates with positively charged His, Arg, or Lys residues at the Xaa position greatly increased. An increase in activity levels against substrates with the aromatic residues Phe and Tyr was also observed. Even when normalizing the activity levels of each protein by designating their activities against the glycine substrate as 100, we observed nearly a 3-fold increase in preference toward the Lys substrate in the S128A protein and a more than 2-fold increase in preference toward the Arg substrate in Y165A. Preferences toward the Tyr and Phe substrates also increased ~3-fold in both enzymes. The results clearly indicate that in addition to limiting the activity levels of SppATk, Ser128 and Tyr165 also play a role in restricting the substrate preference of the enzyme toward peptide substrates.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have performed a detailed site-directed mutagenesis study on an archaeal SPP from T. kodakaraensis. The results have revealed multiple residues that are critical for the peptidase activity of SppATk and provide the first insight into the catalytic mechanism of prokaryotic SPPs. Our analyses strongly indicate that the catalytic center of SppATk is comprised of a Ser-Lys dyad and not the Ser/His/Asp catalytic triad that is present in the majority of serine proteases.

The nucleophilic serine residue of SppATk has been clarified to be Ser162. Along with the inhibitor studies that indicated the protein was a serine peptidase (19), activity was completely abolished in the S162A protein, whereas mutations in the other well conserved serine residues (S128A and S184A) had relatively smaller or no effects on the peptidase activity. Because Ser162 is completely conserved in all of the archaeal SppA homologues as well as the SppA from E. coli and B. subtilis, this serine residue most likely serves as the nucleophile in all prokaryotic SPPs. Besides the three residues examined, no other Ser residue is conserved in the core region of the enzyme, even among the closely related proteins from Thermococcales. Our results also indicate that the residue acting as the general base in SppATk is Lys214. This residue is clearly conserved in the archaeal enzymes from Pyrococcus spp., the methanogens, Thermoplasma spp., Picrophilus, and one homologue from Haloarcula. The residues surrounding the Lys residues are also highly conserved among these proteins, and the sequences can be clearly aligned without any gaps from the nucleophilic Ser162 to a conserved Glu227. Although to a lower extent, the enzyme from Nanoarchaeum equitans also displays similarity and harbors most of these residues. It can therefore be presumed that the residue acting as the general base in these enzymes (Fig. 1, upper group of sequences), corresponds to the Lys214 in SppATk. Although a lysine residue is found in some of the remaining homologues from the halophiles and the Crenarchaeon Pyrobaculum (Fig. 1, lower group of sequences), the relatively lower sequence similarity makes it difficult for us to estimate the general base in these enzymes. Interestingly, the bacterial SppA from B. subtilis also harbors the basic Lys residue, and surrounding sequences are particularly well conserved between the enzyme and SppATk, including the charged residues Lys209, Lys214, Asp215, Arg221, Glu226, and Glu227. This strongly suggests that the bacterial SppA from B. subtilis also utilizes Lys214 as the general base. On the other hand, Lys 214 is not conserved in SppAEc, and Lys residues are not found in the near vicinity. This indicates that the enzyme from E. coli utilizes distinct residues for catalysis.

Besides Ser162and Lys214, which were essential for activity, we found that the S184A, H191A, K209A, D215A, and R221A mutations led to decreases in Vmax or kcat values. The results suggest that these residues, although not essential for activity, may also play a role in the catalytic mechanism. In particular, the mutation of Arg221 had severe effects. Because our results have clearly indicated the presence of a Ser-Lys catalytic dyad in SppATk, one important question to be solved would be how the lysine residue is maintained in a deprotonated state, which would be necessary to increase the nucleophilicity of the serine side chain O{gamma}. The pK' value of the lysine side chain is ~10.8, and the microenvironment surrounding the catalytic center would have to be extremely alkaline. One feasible explanation taking our results into consideration would be that the side chain of Arg221 is positioned in the very near vicinity of the Lys214 side chain. Because the pK' value of an arginine side chain is much higher (~12.5), Arg 221may act to sequester protons that would otherwise tend to protonate Lys214. The positive charge of the protonated Arg side chain may also act to repulse other protons from approaching the Lys214 {epsilon} -amino group. Another possibility would be that Lys214 is surrounded by a neutral or hydrophobic environment, which would also result in an apparent decrease in the pK' value of the {epsilon}-amino group, as reported in LexA (27) and the signal peptidase from E. coli (25, 28).

The S184A mutation also had significant effects on the activity of SppATk. From a comparison of the three-dimensional structures of the bacterial type I signal peptidase (28), Lon protease (29), LexA (27), UmuD' (30), and the {lambda} repressor C-terminal domain (31), which are all Ser/Lys proteases, possibilities of a third residue interacting with the Ser/Lys dyad have been raised (29). The residue is conserved as either a serine or threonine residue, with the side chain O{gamma} forming a hydrogen bond with the catalytic Lys {epsilon}-amino group. Because no other serine or threonine residues are conserved among the SPPs and because the S128A mutation led to an increase in activity, Ser184 may represent the corresponding residue in SppATk. Future studies on the three-dimensional structure of the protein should clarify the roles of Ser184 and Arg221.

As mentioned under "Results," it is intriguing that the replacements of so many highly conserved residues, usually presumed to contribute in maintaining enzyme function, actually lead to increases in peptidase activity. One possibility is that the substrate or the reaction conditions we have applied in our activity measurements differ so much from the actual environment of the enzyme that our results do not reflect at all the actual signal peptide degrading activity of the individual proteins. However, another tempting possibility is that these residues are deliberately present to limit the functional capacity of SppA. In bacteria, SppA is presumed to be anchored to the cell membrane with its catalytic core facing the cytoplasm. Although peptidase activity is necessary for the breakdown of free signal peptides, it should also be important for the cell that the enzyme does not cleave other proteins, whether they are soluble proteins in the cytoplasm or proteins integrated or anchored to the cell membrane. This can be achieved by a variety of strategies on the protein itself or toward its environment. A strict confinement of the substrate specificity of the enzyme would surely lead to a decrease in SPP acting on proteins other than free signal peptides. Limiting the activity levels of the enzyme would also contribute to prevent unintended protein degradation. Preventing the access of SPPs to proteins other than signal peptides would be another practical strategy. Because we have found that the conserved Ser128 and Tyr165 not only place a limitation on activity levels but also act to restrict the substrate specificity of SppATk, the presence of these residues may well be to suppress activity of the enzyme toward unintended substrates.

Although still a minority among the serine proteases, the number of enzymes known to utilize the Ser/Lys catalytic dyad is steadily increasing. In addition to the bacterial type I signal peptidase, Lon protease, LexA, UmuD', and the {lambda} repressor C-terminal domain mentioned above, other examples include the archaeal Lon protease from Methanocaldococcus jannaschii (32), PH1510 from Pyrococcus horikoshii (33), the periplasmic tail-specific protease from E. coli (34), and the C-terminal endoprotease from cyanobacteria (35). This study has revealed that the SPPs from Euryarchaeota are also members of this enzyme family. There are still no indications for a reason why certain serine proteases utilize the Ser/His/Asp catalytic triad, whereas others use the Ser/Lys dyad. Although presumed to execute identical functions, the signal peptidase from the archaeon M. voltae utilizes a catalytic triad (16), whereas the signal peptidase from E. coli harbors a dyad (28). The results of our study also raise the possibility that the SPPs from E. coli and T. kodakaraensis function through distinct catalytic mechanisms. Discovery of novel peptidases/proteases that utilize the Ser/Lys dyad and further structure-function studies on both families of enzymes will be necessary for an understanding of this intriguing distinction in catalytic mechanisms.


   FOOTNOTES
 
* This work was supported by a grant of the National Project on Protein Structural and Functional Analyses 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. Back

1 To whom correspondence should be addressed. Tel.: 81-75-383-2777; Fax: 81-75-383-2778; E-mail: imanaka{at}sbchem.kyoto-u.ac.jp.

2 The abbreviations used are: SPP, signal peptide peptidase; CHES, 2-(cyclohexylamino)ethanesulfonic acid; MCA, {alpha}-4-methyl-coumaryl-7-amide; FRETS, fluorescence resonance energy transfer substrates. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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S. Y. M. Ng, B. Chaban, D. J. VanDyke, and K. F. Jarrell
Archaeal signal peptidases
Microbiology, February 1, 2007; 153(2): 305 - 314.
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