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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Biochemistry. Author manuscript; available in PMC 2010 May 5.
Published in final edited form as:
PMCID: PMC2683473

Functional Identification of Incorrectly Annotated Prolidases from the Amidohydrolase Superfamily of Enzymes


The substrate profiles for two proteins from Caulobacter crescentus CB15 (Cc2672 and Cc3125) and one protein (Sgx9359b) derived from a DNA sequence (gi| 44368820) isolated from the Sargasso Sea were determined using combinatorial libraries of dipeptides and N-acyl derivatives of amino acids. These proteins are members of the amidohydrolase superfamily and are currently misannotated in NCBI as catalyzing the hydrolysis of L-Xaa-L-Pro dipeptides. Cc2672 was shown to catalyze the hydrolysis of L-Xaa-L-Arg/Lys dipeptides and the N-acetyl and N-formyl derivatives of lysine and arginine. This enzyme will also hydrolyze longer peptides that terminate in either lysine or arginine. The N-methyl phosphonate derivative of L-lysine was a potent competitive inhibitor of Cc2672 with a Ki value of 120 nM. Cc3125 was shown to catalyze the hydrolysis of L-Xaa-L-Arg/Lys dipeptides but will not hydrolyze tripeptides or the N-formyl and N-acetyl derivatives of lysine or arginine. The substrate profile for Sgx9359b is similar to that of Cc2672 except that compounds with a C-terminal lysine are not recognized as substrates. The x-ray structure of Sgx9359b was determined to a resolution of 2.3 Å. The protein folds as a (β/α)8-barrel and self associates to form a homo-octamer. The active site is composed of a binuclear metal center similar to that found in phosphotriesterase and dihydroorotase. In one crystal form, arginine was bound adventitiously to the eight active sites within the octamer. The orientation of the arginine in the active site identified the structural determinants for recognition of the α-carboxylate and the positively charged side chains of arginine containing substrates. This information was used to identify 18 other bacterial sequences that possess identical or similar substrate profiles.

In 2008 there were nearly 800 completely sequenced bacterial genomes reported in the NCBI website. A critical assessment of the annotations for the more than 4 million genes contained within these organisms indicates that a significant fraction of the derived enzymes and proteins have an uncertain, incorrect, or ambiguous catalytic function. This observation suggests that substantial parts of the metabolic landscape remain to be identified and that the rules for deciphering catalytic activity from protein sequences and three dimensional structures are quite challenging. Our approach to a limited portion of this difficult problem has been to combine three-dimensional structure determination, computational docking, high throughput screening, and genomic context toward the assignment of function to members of the amidohydrolase superfamily (1-3).

The amidohydrolase superfamily (AHS) is a complex cluster of enzymes that has been shown to catalyze a variety of chemical transformations (4, 5). Well-characterized members of this superfamily include phosphotriesterase (6), urease (7), dihydroorotase (8), and adenosine deaminase (9). The predominant reactions catalyzed by the AHS include isomerizations (10), decarboxylations (11), hydrations (12), and hydrolysis of C-O (13), C-N (14), and P-O (15) bonds. All members of the AHS adopt a (β/α)8-barrel structural fold and contain a mononuclear or binuclear metal center embedded at the C-terminal end of the β-barrel. In vivo, the metal centers can be occupied by Ni2+, Zn2+, Fe2+, or Mn2+ (4). However, certain members of the AHS can be activated by Co2+ and Cd2+ via reconstitution of the apo-enzyme (16). The metal centers found within the active sites of the amidohydrolase superfamily function in catalysis by activating solvent water for nucleophilic attack and/or stabilization of transition state structures (4).

Enzymes within the AHS have been annotated by NCBI as prolidases that presumably catalyze the hydrolysis of dipeptides that contain proline as the C-terminal amino acid. Approximately 2-3% of the known members of the amidohydrolase superfamily have been annotated as prolidases. However, none of these enzymes has been adequately characterized and the breadth of the substrate specificity for variation in the amino acids that can occupy the amino and carboxy termini of potential substrates is unknown. Also unknown are the structural determinants and highly conserved amino acid residues within the active sites of these enzymes that dictate the substrate profile.

In this paper we present the discovery of the catalytic activities for two enzymes of ambiguous function from Caulobacter crescentus CB15 and a related protein derived from an environmental DNA sequence isolated from the Sargasso Sea. C. crescentus naturally inhabits an aquatic environment and plays an important role in biochemical cycling of organic nutrients (17). Based upon amino acid sequence alignments with structurally characterized members of the AHS, it is expected that all three enzymes will contain a binuclear metal center at the active site. The two enzymes from C. crescentus, Cc2672 and Cc3125, are 47% identical in protein sequence to one another and are 47 and 37% identical, respectively, to the enzyme from the Sargasso Sea, Sgx9359b (gi| 44368820). The substrate specificities were determined for these enzymes via the synthesis and characterization of multiple dipeptide libraries containing nearly all possible combinations of L-Xaa-L-Xaa dipeptides using the 20 common amino acids. Contrary to our initial expectations none of the three enzymes was able to hydrolyze dipeptides that contained L-proline at the C-terminus. Cc2672 can hydrolyze dipeptides with either L-lysine or L-arginine at the C-terminus. It is also able to hydrolyze the N-acetyl or N-formyl derivatives of L-lysine or L-arginine and short polypeptides ending in these amino acids. The substrate specificity of Cc3125 is restricted to the hydrolysis of dipeptides with either L-lysine or L-arginine at the C-terminus. The substrate specificity of Sgx9359b is similar to that of Cc2672, except that the C-terminus must be L-arginine. The x-ray structure of Sgx9359b has been determined in the presence of the hydrolysis product, L-arginine. Sgx9359b and Cc2672 are more properly described as carboxypeptidases whereas Cc3125 appears to be a true dipeptide hydrolase.

Materials and Methods


All chemicals were obtained from Sigma or Aldrich, unless otherwise stated. The genomic DNA from C. crescentus CB15 was purchased from the American Type Culture Collection (ATCC). The synthesis of oligonucleotides and DNA sequencing reactions were performed by the Gene Technology Laboratory of Texas A&M University. The pET-30a(+) expression vector was acquired from Novagen. T4 DNA ligase and various restriction enzymes were purchased from New England Biolabs. Platinum Pfx DNA polymerase and the Wizard Plus SV Mini-Prep DNA purification kit were obtained from Invitrogen and Promega, respectively. The N-methyl phosphonate derivative of L-lysine (1) was synthesized according to the method described by Xu et al. and the structure is presented in Scheme 1 (18).

Synthesis of Dipeptide Libraries and Dipeptides

The dipeptide libraries were made as described below. Nineteen preloaded N-Fmoc-protected (or unprotected)-L-amino acid Wang resins (0.02 mmol each of N-Fmoc-L-Ala, N-Fmoc-L-Arg(Mtr), N-Fmoc-L-Asn(Trt), N-Fmoc-L-Asp(OtBu), N-Fmoc-L-Glu(OtBu), N-Fmoc-L-Gln(Trt), N-Fmoc-L-Gly, N-Fmoc-L-His(Trt), N-Fmoc-L-Ile, N-Fmoc-L-Leu, N-Fmoc-L-Lys(Boc), N-Fmoc-L-Met, N-Fmoc-L-Phe, N-Fmoc-L-Pro, N-Fmoc-L-Ser(Trt), N-Fmoc-L-Thr(Trt), N-Fmoc-L-Trp(Boc), N-Fmoc-L-Tyr(tBu), and N-Fmoc-L-Val) and DMF (5 mL) were shaken in a syringe for 30 minutes. The DMF was removed by filtration and then 6 mL of 20% piperidine in DMF was added and the mixture shaken for 30 minutes. This process was repeated. The beads were washed with DMF (4×5 mL) and then N-Fmoc-L-Ala-OH (177.4 mg, 0.57 mmol), HOBt·H2O (87.2 mg, 0.57 mmol), N, N’-diisopropylcarbodiimide (71.8 mg, 0.57 mmol) in DMF (6 mL) was added and shaken overnight. The reagents were removed and the beads was washed with DMF (4 × 5 mL), dichloromethane (4 × 5 mL), methanol (4 × 5 mL) and dried for several hours. To the dried beads was added cocktail R (4 mL, TFA/thioanisole/EDT/anisole (v/v, 90/5/3/2)) and shaken for 3 hours. The N-Fmoc-L-Ala-L-Xaa -amino acids library was obtain after removal of the solvent under reduced pressure, washing with EtOAc/Et2O (v/v, 1/5) and then drying overnight at 50 °C. The library was stirred with 20% piperidine in DMF (5 mL) for 30 minutes to obtain the L-Ala-L-Xaa dipeptide library after removal of the solvent and washing with EtOAc/Et2O (v/v, 1/5). In the same way, the libraries of L-Arg-L-Xaa, L-Asn-L-Xaa, L-Asp-L-Xaa, L-Glu-L-Xaa, L-Gln-L-Xaa, Gly-L-Xaa, L-His-L-Xaa, L-Ile-L-Xaa, L-Leu-L-Xaa, L-Lys-L-Xaa, L-Met-L-Xaa, L-Phe-L-Xaa, L-Pro-L-Xaa, L-Ser-L-Xaa, L-Thr-L-Xaa, L-Trp-L-Xaa, L-Tyr-L-Xaa, and L-Val-L-Xaa libraries were prepared. Mass spectrometry analysis (ESI, positive and negative mode) was used to verify the presence of the 19 members in each of the 19 dipeptide libraries constructed for this investigation.

The dipeptides L-Ala-L-Lys and L-Ala-L-Arg were prepared in a similar fashion using single preloaded N-Fmoc-L-Lys(Boc) and N-Fmoc-L-Arg(Mtr) beads. The tripeptide L-Gly-L-Phe-L-Arg was purchased from Aroz Technologies LLC.

Gene cloning

The genes for Cc2672 and Cc3125 were amplified from the genomic DNA of Caulobacter crescentus CB15. The PCR product for the isolation of the gene for Cc2672 utilized the primers, 5′-AGAACTTCCATATGCGTATGGGGATGAAGATCGCGACGC-3′ and 5′-AGCGAATTCCTACGGGGCCTTCACCACCGCGC-3′. The PCR product for the amplification of the gene for Cc3125 utilized the primer pair, 5′-AGAACTTCCATATGAAACTGCACGTGTTTT GCGTCGCCG-3′ and 5′-ACGGAATTCCTAGTCGTCCTTGACCACGGTCCCG-3′. The PCR products were gel purified, digested with NdeI and EcoRI, ligated to the expression vector pET-30a (+), and then transformed into XL1-blue cells. Individual colonies containing the plasmid were selected on LB plates containing 50 μg/mL kanamycin and then used to inoculate 5 mL of LB. The entire coding region of the plasmids containing the genes for Cc2672 and Cc3125 were sequenced to confirm the fidelity of the PCR amplification. Based upon the reported sequence of gi|44368820 deduced from a DNA sample originally isolated from the Sargasso Sea, the New York SGX Research Center for Structural Genomics (NYSGXRC) cloned and expressed Sgx9359b as a His-tagged protein following codon-optimization and gene synthesis by Codon Devices, Inc (Cambridge, MA). NYSGXRC has made the clone (9359b1BCt7p1) available through the Protein Structure Initiative Material Repository (PSI-MR) at the Harvard Institute of Proteomics (

Protein Purification

Cc2672 and Cc3125 were purified by a combination of gel filtration and ion exchange chromatography. BL21(DE3) Star cells (Novagen) were transformed with a pET-30a(+) plasmid containing either the gene for Cc2672 or Cc3125. A single colony was used to inoculate a 5 mL overnight culture of LB broth containing 50 μg/mL kanamycin. The overnight culture was then used to inoculate 1.0 L of LB medium containing 50 μg/mL kanamycin. The culture was grown at 30 °C, and induced by the addition of 0.5 mM isopropyl-D-thiogalactopyranoside (IPTG) after an A600 of 0.6 had been reached. At this time 1.0 mM Zn(OAc)2 was added to the culture. Cells were harvested by centrifugation (5000 rpm for 15 minutes) 18 hours after IPTG induction and stored at -80 °C. For the purification of Cc2672, the frozen cells were resuspended in 50 mM Hepes, pH 8.0, and lysed by sonication (5 second pulses for 30 minutes) at 0 °C. After centrifugation, the nucleic acids were removed by adding 2% (w/v) protamine sulfate dropwise. The supernatant solution after centrifugation was fractionated using ammonium sulfate. The precipitated protein (60-75% ammonium sulfate saturation) was resuspended in a minimum volume of 50 mM Hepes, pH 8.0, and loaded onto a Superdex 200 gel filtration column (Amersham Pharmacia) and eluted at a flow rate of 2.0 mL/minute. The fractions containing the desired protein were pooled based on the results from SDS-PAGE. The protein was loaded onto a Resource Q anion exchange column (Amersham Pharmacia) and eluted with a linear gradient of NaCl in 20 mM Hepes, pH 8.0. The purity of the protein was verified by SDS-PAGE. The protein Cc3125 was purified in a manner similar to Cc2672 except that the protein was initially fractionated with ammonium sulfate between 0 and 65% of saturation. Cc3125 was eluted from the Resource Q column at approximately 70 mM NaCl.

For purification of Sgx9359b, E. coli BL21(DE3) Star cells were transformed with the plasmid encoding the gene for this protein. One liter of LB was inoculated with a 5 mL overnight culture. The inoculated culture was grown with agitation at 30 °C to an OD600 of 0.6, supplemented with 1.0 mM Zn(OAc)2, induced with 0.5 mM IPTG, and then allowed to grow at 30 °C for 14 hours. The pellet was harvested by centrifugation and suspended in binding buffer (20 mM Hepes, pH7.9, 0.5 M NaCl and 5 mM imidazole). The cells were disrupted by sonication and the insoluble debris was removed by centrifugation (15 minutes at 10,000 rpm). The clarified cell extract was applied to a 24 mL column of chelating Sepharose Fast Flow (Amersham Biosciences) charged with Ni2+ and precalibrated with binding buffer. The column was washed with 1000 mL of binding buffer until the absorbance of the flow-through at 280 nm was constant. The His-tagged Sgx9359b protein was eluted with a gradient of elution buffer (10 mM Hepes, pH7.9, 0.25 M NaCl and 0.5 M imidazole). The protein thus obtained was further purified by a Resource Q anion exchange column after NaCl was removed by dialysis. Sgx9359b was eluted from the column with a gradient of NaCl in 20 mM Hepes, pH 8.0. The protein was >95% pure based upon SDS-PAGE.

Amino Acid Sequence and Metal Content

The purified proteins Cc2672 and Cc3125 were subjected to N-terminal amino acid sequence analysis by the Protein Chemistry Laboratory at Texas A&M University. The N-terminal amino acid sequence analysis for the first 11 amino acids of Cc2672 gave the sequence as AEIKAVSAARL. This result indicates that the protein was either initially expressed from amino acid 27 or that the first 26 amino acids were lost to proteolysis. For Cc3125 the first six amino acid residues had the sequence QVSYVR. This result indicates that the first 21 amino acid residues were lost to proteolysis. The metal content of the purified proteins was determined with a Perkin-Elmer Analyst 700 atomic absorption spectrometer and by inductively coupled plasma emission-mass spectrometry (ICP-MS). Cc2672 contained 2.0 equivalents of Zn2+ and Cc3125 contained an average of 0.7 equivalents of Zn2+. Sgx9359b did not contain any metal after completion of the initial purification. The active site was reconstituted with metal by adding 2 equivalents of ZnCl2 and 10 mM potassium bicarbonate overnight in 50 mM Hepes, pH 7.5, to the purified Sgx9359. The protein was subsequently passed through a PD-10 desalting column equilibrated with metal-free 50 mM Hepes, pH 7.5, to remove loosely bound Zn2+ ions. The reconstituted protein was found to contain 1.8 equivalents of Zn2+ as determined by ICP-MS.

Peptidase Activity Measurements

A modification of a ninhydrin-based colorimetric assay was adopted for the peptidase activity of Cc2672, Cc3125 and Sgx9359b (19). The Cd-ninhydrin reagent solution was prepared by dissolving 0.4 g of ninhydrin into 40 mL of 99.5% ethanol and 5 mL of acetic acid. To this solution was added 0.5 g of CdCl2 dissolved in 0.5 mL of water. The peptide hydrolysis reactions were conducted at 30 °C in 50 mM Hepes, pH 8.0. Routinely, 120 μL of the reaction solution and 240 μL of the Cd-ninhydrin reagent were mixed and then heated in a 96-well block for 5 minutes in an 80 °C water bath. After cooling to room temperature, 250 μL of the reaction solution from each well was transferred to a 96-well UV-visible microplate, and the absorbance at 507 nm was recorded with a SPECTRAmax plate reader from Molecular Devices. The measurement of the hydrolysis of N-formyl- and N-acetyl-L-amino acid derivatives was performed using the same procedure. A quantitative analysis of the liberated amino acids was conducted by the Protein Chemistry Lab at Texas A&M University.

Screening of Dipeptide Libraries

Each of the L-Xaa-L-Xaa dipeptide libraries consisted of a mixture of dipeptides with an identical L-amino acid at the N-terminus but 19 different amino acids at the C-terminus (L-cysteine was not included in any of the libraries). A total of 19 dipeptide libraries containing 361 different dipeptides were used to screen the dipeptidase activity of Cc2672, Cc03125 and Sgx9359b. For each dipeptide library, 120 μL of a reaction mixture containing ~0.1 mM of each dipeptide in 50 mM Hepes, pH 8.0, was incubated at 30 °C for variable lengths of time with multiple concentrations of enzyme. The reactions were quenched by adding 240 μL of the ninhydrin reagent and then the samples were incubated for 5 minutes at 80 °C before rapid cooling to room temperature. For each dipeptide library, 10 to 1000 nM of enzyme were used in the initial screening assays with each dipeptide library. Control reactions with no enzyme or added substrate were conducted simultaneously. The hydrolytic rates for the enzymatic turnover of the 19 dipeptide libraries were determined based on the change in absorbance at 507 nm after subtraction of the background for the two control reactions. The relative rates were determined by a fit of the data to equation 1, where y is the change in absorbance at 507 nm, x is concentration of enzyme and b is the relative rate constant. These experiments measure the rate of formation of free amino acids within the entire library. In these measurements we have elected to vary the enzyme concentration for a fixed period of time rather than vary the time at a fixed enzyme concentration.


Amino Acid Analysis

HPLC methods were employed to identify the specific amino acids that were released from the dipeptide libraries after the addition of enzyme. Selected dipeptide libraries were used to determine the substrate specificity for each of the three enzymes under study. The hydrolysis reactions were conducted in 25 mM ammonium bicarbonate buffer, pH 8.0. For each dipeptide library, variable amounts of enzyme (1-1000 nM) were used to quantify the products for each enzyme reaction. Two dipeptide libraries (L-Ala-L-Xaa and L-Phe-L-Xaa) were assayed with Cc2672, two dipeptide libraries (L-Met-L-Xaa and L-Leu-L-Xaa ) were used for Cc31250, and two dipeptide libraries (L-Ala-L-Xaa and L-Leu-L-Xaa) for Sgx9359b. Routinely, 30 μL of a reaction mixture containing variable concentrations of enzyme and ~0.1 mM of each dipeptide in a 96-well block were incubated at 30°for 2 hours (in the case of Cc2672) or overnight (in the case of Cc3125 and Sgx9359b). A 25 μL aliquot was removed from each reaction and diluted to 200 μL with water. The enzyme was removed by filtration through an Amicon centrifuge filter column (Ultracel YM-10). The samples were dried under reduced pressure and then reconstituted with water prior to quantitative amino acid analysis. Relative reaction rates were determined by fitting the change in amino acid concentration as a function of enzyme concentration according to equation 1.

Kinetic Measurements for Individual Substrates

Selected peptides, N-formyl-L-Arg, N-formyl-L-Lys, N-acetyl-L-Arg, and N-acetyl-L-Lys were used as substrates to measure the kinetic parameters for Cc2672, Cc3125 and Sgx9359b. The kinetic parameters, kcat , Km and kcat/Km were determined using the ninhydrin assay in 50 mM Hepes, pH 8.0. The kinetic constants were determined by fitting the initial velocity data to equation 2, where ν is the initial velocity, kcat is the turnover number, Et is the enzyme concentration, A is the substrate concentration, and Km is the Michaelis constant. Data conforming to competitive inhibition were fit to equation 3 where Ki is the competitive inhibition constant and the other constants have been previously defined.


Structure Determination and Refinement

Diffraction quality crystals of Sgx9359b were obtained by sitting drop vapor diffusion after mixing 1 μL of protein solution (21 mg/mL, containing 10% glycerol) with 1 μL of mother liquor composed of 100 mM succinic acid, pH 7.0, and 13-17% of PEG3350. Rod-shaped crystals appeared in 3-4 days and continued to grow for 2-3 more days. Protein crystals were soaked for 1-10 minutes in mother liquor supplemented with 15-30% glycerol as a cryoprotectant. To obtain a Zn-bound complex, crystals were soaked in cryoprotectant supplemented with 5 mM zinc sulfate for 5-10 minutes before freezing in liquid nitrogen. X-ray diffraction data were collected from crystals cooled in a nitrogen stream (100 K) at the National Synchrotron Light Source beamline X29A (Brookhaven National Lab, Upton, NY). Data were indexed, integrated and scaled with the HKL2000 software package (20). Statistics are provided in the Table 1.

Table 1
Data collection and refinement statistics for Sgx9359b

The crystal structures were determined by molecular replacement using MOLREP (CCP4 package suit) and the PDB file 2R8C as the search model (21). The Zn-free and Zn-substituted crystals were isomorphous and exhibited diffraction consistent with the orthorhombic space group P212121. The asymmetric unit contains 8 protein monomers arranged as a homo-octamer with 422 point symmetry, allowing for the application of non-crystallographic symmetry restraints (NCS) throughout refinement. Models were refined with REFMAC 5.3 (CCP4 package suit) (22). The Zn-free structure was refined to a resolution of 2.3 Å, with Rwork and Rfree of 22.6% and 28.0%, respectively, and the Zn-bound structure was refined to a resolution of 2.62 Å with Rwork and Rfree of 23.0% and 26.3%, respectively (Table 1). All interactive model building was performed with COOT (23); the stereochemistry of the models was verified by PROCHECK (24).

CLUSTAW ( was used for determining sequence alignments. Rendering of sequence alignments was performed with ESpript 2.2 http// Molecular modeling was performed using CPHmodels on-line software ( and the ligands (arginine or lysine) were fitted manually. Figures were prepared with PyMOL (25) and Adobe Photoshop 7.0.

Differential Scanning Fluorimetry (ThermoFluor)

Fluorescence-monitored thermal denaturation was performed with 20 μL samples per well in a 96-well plate format at a protein concentration of 10 μM in the presence of SYPRO Orange (Invitrogen), screening buffer (100 mM Hepes, pH 7.5, and 150 mM NaCl) and an arginine concentration range of 1-2500 μM (26). The 96-well plate was sealed with Optical-Quality Sealing Tape (Bio-Rad) and the temperature ramped in an iCycler iQ Real Time PCR System (Bio-Rad) over a range of 20 to 95 °C in 1 °C increments. The excitation and emission wavelengths were 490 and 575 nm, respectively. Changes in fluorescence intensity were monitored by the iCycler’s internal CCD camera and the data were fit to a two state-transition to determine Tm.


Purification and Properties of Cc2672, Cc3125 and Sgx9359b

The genes for Cc2672 and Cc3125 were amplified from C. crescentus CB15 and cloned into pET-30a(+) for expression in E. coli. The protein Cc2672 expressed quite well when the plasmid harboring the gene for Cc2672 was transformed into BL21(DE3) Star cells. The protein was purified to homogeneity and found to contain 2.0 equivalents of Zn2+ per subunit. The purified protein gave a single band on SDS-PAGE with an apparent molecular weight of 43 kDa, which is slightly smaller than the theoretical molecular weight of 45 kDa calculated from the reported gene sequence. The isolated enzyme was missing 26 amino acid residues from the N-terminus. The protein Cc3125 was purified using a nearly identical protocol as for the isolation of Cc2672. However, the yield of the isolated protein was lower due to poor solubility. The purified protein contained an average of 0.7 equivalents of Zn2+ per subunit and was missing the first 21 amino acids from the N-terminus. After the initial purification of Sgx9359b, it was determined that no metals were bound to the protein. However, the protein was reconstituted with Zn2+ and 1.8 equivalents of Zn2+ were bound to the protein per subunit after overnight incubation with 10 mM bicarbonate and Zn2+.

Screening of Dipeptide Libraries

Nineteen dipeptide libraries were used to screen the dipeptidase activity of Cc2672, Cc3125 and Sgx9359b using the ninhydrin assay for measurement of free amino acids. The relative preferences for the first amino acid residue within each of these dipeptide libraries for the three proteins were determined and illustrated in Figures 1a-c. These experiments measure the average rate of formation of free amino acids within each of the dipeptide libraries. All three proteins are promiscuous for the amino acid at the N-terminus but the relative rates of hydrolysis vary within each of the dipeptide libraries. Cc2672 shows the highest activity with the L-Trp-L-Xaa library whereas Cc3125 shows the most pronounced activity with the L-Leu-L-Xaa library. The L-Phe-L-Xaa library was the best substrate for Sgx9359b. All three proteins exhibit very low activity with the L-Ile-L-Xaa, L-Asp-L-Xaa and L-Glu-L-Xaa libraries.

Figure 1
Relative substrate activities for the hydrolysis of 19 dipeptide libraries of the type L-Xaa-L-Xaa where a single amino acid is at the N-terminus (defined on the x-axis) and 19 different amino acids were at the C-terminus. (A) Cc2672; (B) Cc3125; and ...

Substrate Specificity of Cc2672, Cc3125 and Sgx9359b

Amino acid analysis was used to determine the relative rates of hydrolysis for specific dipeptides within selected dipeptide libraries. Cc2672 was assayed with the L-Ala-L-Xaa and L-Phe-L-Xaa dipeptide libraries. The amino acids detected from the hydrolysis of the L-Ala-L-Xaa library were limited to alanine, arginine, and lysine. The amino acid products detected from the hydrolysis of the L-Phe-L-Xaa library were limited to phenylalanine, arginine, and lysine. Since alanine and phenylalanine were only detected when libraries containing these amino acids were at the N-terminus, the substrate specificity of Cc2672 at the C-terminus is limited to arginine and lysine. All of the other dipeptides libraries have not been tested in this manner. However, based upon the finding of only arginine and lysine residues from the L-Ala-L-Xaa and L-Phe-L-Xaa libraries we assume that all of the other dipeptide libraries will exhibit the same specificity for the C-terminus. Under these reaction conditions the catalytic activity of Cc2672 for L-Xaa-L-Arg is approximately twice that of L-Xaa-L-Lys. The relative reaction rates are presented in Figure 2a. The relative rates of hydrolysis for the other dipeptides in these libraries are less than 1%.

Figure 2
Amino acid analysis after the hydrolysis of selected dipeptide libraries by Cc2672, Cc3125 and Sgx9359b. (A) Cc2672 with L-Ala-L-Xaa (black) and L-Phe-L-Xaa (grey); (B) Cc3125 with L-Met-L-Xaa (black) and L-Leu-L-Xaa (grey); (C) Sgx9359b with L-Ala-L ...

The enzyme Cc3125 was assayed with the dipeptide libraries L-Met-L-Xaa and L-Leu-L-Xaa. With the L-Met-L-Xaa library only arginine, lysine, and methionine were detected after amino acid analysis. When the L-Leu-L-Xaa library was used as a substrate only arginine, lysine and leucine were identified as the reaction products. Therefore, this enzyme is specific for the release of arginine and lysine from the C-terminus of dipeptides. The relative rates are illustrated in Figure 2b. Under these reaction conditions Cc3125 exhibited a higher activity with L-Leu-L-Lys than for L-Leu-L-Arg but with the L-Met-L-Xaa library the relative rates were approximately the same. The upper limit for the hydrolysis of other amino acids at the C-terminus is less than 1%.

The Sgx9359b enzyme was assayed with the L-Ala-L-Xaa and L-Leu-L-Xaa dipeptide libraries. With the L-Ala-L-Xaa library only alanine and arginine were identified as free amino acids whereas with the L-Leu-L-Xaa library only leucine and arginine were found. Therefore, this enzyme is specific for the hydrolysis of L-Xaa-L-Arg dipeptides. The upper limit for the hydrolysis of L-Ala-L-Lys relative to the hydrolysis of L-Ala-L-Arg is 1%. The relative rates for the hydrolysis of the two libraries are shown in Figure 2c.

Deacetylase and Tripeptidase Activity

Cc2672 was tested for the ability to hydrolyze other substituted amino acid derivatives. The enzyme was tested with 19 N-formyl-L-Xaa and N-acetyl-L-Xaa (except L-cysteine) derivatives. The only compounds hydrolyzed were the N-formyl- and N-acetyl derivatives of L-lysine and L-arginine. Cc2672 was also tested with two tripeptides L-Gly-L-Phe-L-Arg and L-Gly-L-Ala-L-Tyr. This enzyme was unable to hydrolyze L-Gly-L-Ala-L-Tyr but it was able to hydrolyze the terminal L-arginine from L-Gly-L-Phe-L-Arg. Cc3125 was unable to hydrolyze any of the N-formyl- or N-acetyl-L-amino acid derivatives. It was also not able to hydrolyze the L-Gly-L-Phe-L-Arg tripeptide. Sgx9359b was found to hydrolyze N-formyl-L-Arg, N-acetyl-L-Arg and L-Gly-L-Phe-L-Arg but does not exhibit any hydrolytic activity when the C-terminal amino acid is any amino acid other than L-arginine.

Kinetic Constants for Cc2672, Cc3125 and Sgx9359b

The kinetic constants for the three enzymes were tested with L-Ala-L-Arg, L-Ala-L-Lys, N-formyl-L-Arg, N-formyl-L-Lys, N-acetyl-L-Arg and N-acetyl-L-Lys. Also tested was the tripeptide L-Gly-L-Phe-L-Arg. The kinetic data were fit to equation 2 and the catalytic constants are presented in Table 2. The N-methyl phosphonate derivative of L-lysine (1) was tested as an inhibitor for the hydrolysis of N-acetyl-L-lysine by the enzyme Cc2672. It was a potent competitive inhibitor with a Ki value of 120 nM from a fit of the data to equation 3.

Table 2
Kinetic parameters of Cc2672, Cc3125 and Sgx9359ba


The purified Sgx9359b exhibited a clean two-state melting transition (Figure 3), characterized by a Tm of 47.6 °C. A significant increase in Tm (i.e., greater than 10 °C stabilization) of was observed at arginine concentrations of 25 μM or higher. This stabilizing effect appeared to plateau at approximately 500 μM arginine (data not shown).

Figure 3
Thermofluor analysis for the stabilization of Sgx9359b by added arginine. The concentrations of arginine are as follows: (●), 0 μM; (■), 25 μM; ([diamond]), 500 μM; ([triangle]), 2500 μM. Additional details ...

Structure of Sgx9359b

The crystal structure of Sgx9359b reveals a homo-octamer in the asymmetric unit (Figure 4), which buries in excess of 30,000 Å2 of accessible surface area, suggesting that this represents the physiologically relevant assembly. Consistent with this assignment, gel-filtration chromatography indicates that Sgx9359b has an apparent molecular weight of over 250,000 (data not shown). The two structures, Zn-free (PDB code: 3BE7) and Zn-bound (PDB code: 3DUG) are nearly identical with RMSD (root mean square deviation) less than 0.3 Å for all Cα. These structures can be described as two tightly associated tetramers related by two-fold rotational symmetry with overall 422 point symmetry (Figure 4). Each protein monomer consists of two structural domains, the catalytic TIM-barrel domain and a smaller β-barrel domain (see Figure 4b and Figure 5). The beta-barrel domain is composed of an outer layer formed by the extreme N-terminal segment (residues 1-55) and an inner layer formed by the extreme C-terminal segment (residues 358-398) of each monomer. The former is exposed to solvent and is the most flexible part of the molecule, with relatively weak electron density and high B-factors. In contrast, the residues of the inner layer are well ordered, being sandwiched between the outer layer of the β-barrel domain and the TIM-barrel domain.

Figure 4
Conformation of Sgx9359b octamer. (A) The space-filled model of Sgx9359b as a homo-octamer composed of two identical tetramers related by C2-fold symmetry. (B) The same model shown as a cartoon with Zn and bound arginine atoms drawn as spheres. (C) The ...
Figure 5
Conformation of the Sgx9359b monomer is drawn as a cartoon model. The catalytic TIM-barrel domain is painted in blue (alpha-helices) and yellow (beta-sheets). The side chains of active site residues are shown as sticks and the two zinc atoms are shown ...

Within the octamer, each monomer makes contacts with four other monomers. This arrangement separates the catalytic sites by ~ 40 Å and notably each catalytic site is formed exclusively by residues contributed by a single monomer (Figures 4b and 4d). A particularly interesting feature is depicted in Figures 4c and 4d, where two adjacent monomers are inter-connected by extended loops, resulting in the formation of a single narrow continuous entrance that provides access to both active sites. This “pseudo-dimer” consists of monomers related by a local two-fold symmetry, with four such pseudo-dimers per octamer.

The catalytic domain is a typical TIM-barrel structure (Figure 5), composed of 8 inner β-strands surrounded by 11 outer alpha helices. A single binuclear zinc-binding site is composed of six conserved residues, including four histidines (His-62, His-64, His-223, His-243), Asp-315 and Lys-182. The latter is carboxylated in the presence of Zn but appears unmodified in the absence of Zn (see below). The zinc binding site is located in a cavity formed by one short and two longer loops connecting α7 and α8, βF and α1, and βH and α3 , respectively. The short loop is relatively flexible and solvent accessible, while the two long loops are rigid due to numerous interactions along the octamer interface. Openings between the loops create access to the active site.

Active Site of Sgx9359b

In the process of refining the Zn-free structure, residual electron density that could not be attributed to protein persisted near the putative Zn-binding site in all eight monomers, with subunit A exhibiting the most prominent features. This density is well accommodated by arginine, which also affords a highly plausible hydrogen bonding network (Figures 6a and 6b). As arginine was not added to either the purified protein or to the crystallization buffers, this ligand must possess sufficiently high affinity to be retained during purification and subsequent crystallization. The high complementarity of arginine to its binding site leaves little space for solvent; only two water molecules are located in the vicinity of the guanidine group, although there are no direct interactions. In the A monomer, weak density is present that is suggestive of a short peptide containing a C-terminal arginine.

Figure 6
(A) 2FoFc electron density map (sigma cutoff 1.0) drawn around residues at the active site of a Zn-free Sgx9359b monomer. Arginine atoms (C-carbons are pink) were omitted from map calculations. (B) Similar to panel A where polar interactions between ligand ...

The Zn-soaked structure at 2.62 Å exhibited two very prominent features, modeled as Zn atoms, in the active site (Figure 6c); strong corresponding features (greater than 10 σ) were also present in anomalous difference Fourier maps (not shown). The refined occupancies for the two Zn atoms are slightly different. Znα is coordinated by the side chains of His-62, His-64 and Asp-315, and refined to an occupancy close to 1.0. Znβ is coordinated by the side chains of His-223, His-243 and the carboxylated Lys-182, and refined to an occupancy of about 0.8. Additional Zn-binding sites were identified in each monomer, but are located on the protein surface and are distant from the active site. It is notable that a soak of 5 minutes in 5.0 mM zinc sulfate was sufficient for almost complete carboxylation of Lys-182. In the Zn-free structure, Lys-182 was unmodified and likely adopts at least two alternate conformations (Figure 6d). Interestingly, the replacement of water by zinc does not change the overall conformation of the active site, and the relative orientations of bound arginines and hydrogen bond patterns are quite similar in both the Zn-free and Zn-bound structures.


Three enzymes with significant sequence identity to one another from the amidohydrolase superfamily have been purified and their substrate profiles determined for the first time. The genes for Cc2672 and Cc3125 were cloned from C. crescentus and the gene for Sgx9359b was chemically synthesized from a DNA sequence originally harvested from the Sargasso Sea. All three enzymes have previously been annotated as L-Xaa-L-Pro dipeptidases but a literature lineage to this postulated catalytic activity is obscure. These enzymes have now been found experimentally to catalyze the hydrolysis of dipeptides but none of them is able to hydrolyze an L-Xaa-L-Pro dipeptide and thus the current biochemical annotations for these three enzymes are incorrect. The substrate specificities for the three proteins are similar but not identical.

Cc2672 is able to hydrolyze dipeptides that contain either an L-arginine or L-lysine at the C-terminus. The best substrate determined thus far is L-Ala-L-Arg with a kcat of 44 s-1 and a kcat/Km of 105 M-1 s-1. In addition, this enzyme is also able to hydrolyze the N-acetyl and N-formyl derivatives of L-lysine and L-arginine and longer peptides that terminate in amino acids with a positively charged functional group. Thus, the substrate specificity is more correctly annotated as a carboxypeptidase with a requirement for a C-terminal L-arginine or L-lysine. The substrate specificity of Cc3125 is more restrictive in that only dipeptides terminating in either L-lysine or L-arginine are hydrolyzed. Simple N-acyl derivatives of these amino acids are not substrates nor are longer peptides hydrolyzed. The best substrate determined thus far is L-Ala-L-Lys with a kcat of 5 s-1 and a kcat/Km of 5 × 104 M-1 s-1. Compounds terminating in lysine are somewhat better substrates than those terminating in arginine.

The substrate profile for Sgx9359b is similar in many respects to that of Cc2672. However, the C-terminal residue is restricted to L-arginine and it will not catalyze the hydrolysis of compounds that terminate in L-lysine. Dipeptides are hydrolyzed with a broad tolerance for changes in the amino acid at the N-terminus. Tripeptides are also hydrolyzed in addition to the N-acetyl and N-formyl derivatives of L-arginine. The best substrate discovered thus far is L-Ala-L-Arg with a kcat of 3 s-1 and a kcat/Km of 1 × 104 M-1 s-1.

Sgx9359b is the only one of these three proteins to be successfully crystallized and its three dimensional structure determined. Other proteases of different specificities share some structural similarity to Sgx9359b. For example, iso-aspartyl dipeptidase (IAD) from E. coli has a TIM-barrel domain with a binuclear Zn-binding site, and it also forms a homo-octamer with considerable similarity to Sgx9359b (27). Quite remarkably, the Sgx9359b protein that was expressed and purified from E. coli was found to contain eight arginines bound to the eight catalytic sites of the octamer. This retention of ligand and the substrate specificity of Sgx9359b are consistent with the ThermoFluor results demonstrating significant stabilization of the protein in the presence of low concentrations of arginine.

The structure of Sgx9359b with arginine bound to the active site as a product complex has revealed those amino acids in the active site that help to define the substrate specificity for these three enzymes. The guanidinium group of arginine is ion-paired to the side chain carboxylates of Glu-289 and Asp-265. Asp-265 is found at the end of β-strand 7 and is conserved in all three enzymes examined in this paper. Glu-289 is located in the loop that follows β-strand 7. In Cc2672 this residue is an aspartate whereas in Cc3125 this residue is an asparagine. It would thus appear that the aspartate at the end of β-strand 7 is more important for recognition of the positively charged side chains of lysine and arginine. The α-carboxylate of the arginine product is ion-paired with the side chain imidazole groups of His-142 and His-225. His-142 is found in the loop that follows β-strand 3 and His-225 is found at the end of β-strand 5. The histidine at the end of β-strand 5 is conserved in all three proteins but the histidine in the loop that follows β-strand 3 is not conserved in Cc3125. This would suggest that the histidine after β-strand 5 is more significant.

The α-amino group of the bound arginine in the active site of Sgx9359b is 3.2 Å away from the side chain carboxylate of Asp-315 and 3.3 Å away from the β-metal ion. Asp-315 is an invariant residue in all members of the amidohydrolase superfamily at end of β-strand 8 that also ligates the α-metal ion. The position of this residue is consistent with previous proposals (28) that this aspartate is critical for the transfer of a proton from the attacking hydroxide or water to the leaving group (in this case the α-amino group of arginine).

Molecular Modeling of Cc2672 and Cc3125

All three proteins studied in this investigation share high amino acid sequence identities among the catalytic site residues. The sequence alignment is presented in Figure 7. This alignment has enabled the application of molecular modeling on the basis of the Sgx9359b structures to rationalize the overlapping yet distinct substrate preferences. As seen in Figure 8, hydrogen bond networks around the active sites of all three proteins look very similar with very few exceptions. The binuclear Zn-binding site consists of 4 histidines, one aspartate and one lysine. The arginine-interacting residues are almost identical, with one exception. The side chain from Glu-289 maintains two hydrogen bonds with the guanidinium group of arginine in the Sgx9359b structure. From the sequence alignment (Figure 7) and molecular modeling data (Figure 8) Cc2672 has Asp-318, and Cc3125 has Asn-320 in this position, although the adjacent residues are identical for all three proteins. Another important feature of the ligand-binding site is that the Sgx9359b structure has only four polar amino acid side chains in the vicinity of the guanidinium group (Asp-265, Ser-269, Glu-289, and Gln-298), three of which are in direct interactions with the ligand. By contrast, the remaining residues inside the active site pocket are hydrophobic (Figure 8), including Val-268, Ile-272 and Leu-273 adjacent to arginine-interacting residues. Attempts to dock lysine in the active site of Sgx9359b results in an unsatisfied hydrogen bond acceptor (Figure 9), suggesting a plausible explanation for the observed substrate specificity. In contrast, the Cc2672 counterparts of the hydrophobic residues in Sgx9359b are replaced by more polar residues (Figures 77 and and8),8), such as Asn-297, Thr-301 and Gln-302. As a result, the Cc2672 catalytic site has a more complex hydrogen bond network with all hydrogen bond donors and acceptors satisfied even in the absence of bound arginine or lysine. For example, Asp-294 may be hydrogen-bonded to His-90 and Asn-297, Asp-318 is hydrogen-bonded to Gln-302, Thr-298 and Thr-301, and the carbonyl oxygen of Gln-325 is connected to water. The extensive hydrogen bond network may create more flexibility that results in an equal opportunity for both arginine and lysine to bind into the active site of Cc2672. The same can be concluded about the active site of Cc3125 which is almost 80% identical to that of Cc2672, thus explaining their overlapping substrate specificities. One important difference between these two enzymes is that Cc3125 has Asn-320 instead of Asp-318 and Asp-304 instead of Gln-302. These pairs of residues are in close vicinity to one another and most likely their side chains maintain hydrogen bond contact. However, since Asn-320 has only one hydrogen bond acceptor, the binding mode of the guanidino group of arginine will likely be different from that found in the Sgx9359b structure (Figure 8).

Figure 7
Sequence alignment of Sgx9359b, Cc2672 and Cc3125. The zinc binding residues are marked by filled triangles and the arginine-binding residues are marked with dots. The Sgx9359b sequence is labeled according to the protein database entry EAI78147. The ...
Figure 8
(A) Scheme of polar interactions between bound arginine and protein in the active site of Sgx9359b. Hydrogen bonds (distance cutoff 3.2 Å) are shown as dashes. Schemes of polar interactions at the active sites of Cc3135 (panel B) and Cc2672 (panels ...
Figure 9
Fitting lysine as a ligand at the active site of Sgx9359b most likely leaves at least one hydrogen-bond acceptor unsatisfied (shown by arrows). This may explain why this protein prefers arginine as a C-terminal residue of a substrate.

The discovery of function for these three enzymes represents the first demonstration of dipeptidase or carboxypeptidase activity for members of the amidohydrolase superfamily where the terminal amino acid in the substrate is restricted to either L-arginine and/or L-lysine. The critical residues located within the active sites of these three enzymes were used to identify other enzymes within the AHS with similar or identical catalytic functions. In particular, the specific residues that were shown to be involved in recognition of the side chain for either arginine or lysine at the C-terminus and the α-carboxylate of substrates were incorporated into the search model. For the three proteins characterized in this paper, the second histidine after β-strand 5 and the aspartate after the end of β-strand 7 are invariant (see Figure 7). A search of the non-redundant protein sequences in NCBI via a simple BLAST protocol identified 18 other sequences that are homologous to Cc2672, Cc3125, and Sgx9359b. A dendrogram that identifies these sequences is presented in Figure 10. Of these protein sequences, 3 of them, Sala0935 (from Sphingopyxis alaskensis RB2256), Swit2115 (from Sphinomonas wittichii RW1), and Saro2823 (from Novosphinogobium aromaticivorans DSN 12444) are more closely related to Cc3125. The remaining 15 sequences are closer to Cc2672 and Sgx9359b. In these sequences the histidine in the loop after β-strand 3 and the glutamate/aspartate in the loop after β-strand 7 are also fully conserved. All of these proteins are expected to catalyze the hydrolysis of N-substituted derivatives of L-lysine and/or L-arginine.

Figure 10
Dendrogram of bacterial homologues to Cc2672, Cc3125, and Sgx9359b. The gi numbers for the proteins in this dendrogram are as follows: Sala0935 (gi|103486425), Cc3125 (gi|16127355), Swit2115 (gi|148555030), Saro2823 (gi|87200836), Acid345_3982 (gi|94971008), ...

A BLAST search against the C. crescentus genomic database shows that in addition to Cc2672 and Cc3125 there is at least one other protein that is closely related to Sgx9359b. This protein Cc0300 is 38% identical in sequence to Sgx9359b. A sequence alignment (not shown) also demonstrated that all three proteins from C. crescentus are closely related. However, all of the residues from Cc0300 that potentially involve the binding of the C-terminal residue of substrates are hydrophobic suggesting that Cc0300 is a probable carboxypeptidase with a specificity towards hydrophobic residues at the C-terminus.


amidohydrolase superfamily
American Type Culture Collection
isopropyl thiogalactopyranoside
root mean squared deviation
iso-aspartyl dipeptidase


This work was supported in part by the NIH (GM071790 and GM074945). The X-ray coordinates and structure factors for Sgx9359b have been deposited in the Protein Data Bank (PDB accession codes: 3BE7 and 3DUG).


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