Massively parallel, computationally guided design of a proenzyme.

Confining the activity of a designed protein to a specific microenvironment would have broad-ranging applications, such as enabling cell type-specific therapeutic action by enzymes while avoiding off-target effects. While many natural enzymes are synthesized as inactive zymogens that can be activated by proteolysis, it has been challenging to redesign any chosen enzyme to be similarly stimulus responsive. Here, we develop a massively parallel computational design, screening, and next-generation sequencing-based approach for proenzyme design. For a model system, we employ carboxypeptidase G2 (CPG2), a clinically approved enzyme that has applications in both the treatment of cancer and controlling drug toxicity. Detailed kinetic characterization of the most effectively designed variants shows that they are inhibited by ∼80% compared to the unmodified protein, and their activity is fully restored following incubation with site-specific proteases. Introducing disulfide bonds between the pro- and catalytic domains based on the design models increases the degree of inhibition to 98% but decreases the degree of restoration of activity by proteolysis. A selected disulfide-containing proenzyme exhibits significantly lower activity relative to the fully activated enzyme when evaluated in cell culture. Structural and thermodynamic characterization provides detailed insights into the prodomain binding and inhibition mechanisms. The described methodology is general and could enable the design of a variety of proproteins with precise spatial regulation.

Production of Long-Acting CNGRC-CPG2 Fusion Proteins: New Derivatives to Overcome Drug Immunogenicity of Ligand-Directed Enzyme Prodrug Therapy for Targeted Cancer Treatment.

Objectives: Aminopeptidase N (APN) is an enzyme highly expressed in metastatic cancers and could be used in targeted cancer therapy. Our previous work showed the successful construction of CNGRC-carboxypeptidase G2 (CPG2) and CNGRC-CPG2-CNGRC fusion proteins. Our conjugates and prodrugs were effective in targeting high APN-expressing cancer cells. In the present study, we aim to produce long-acting fusion proteins to overcome 2 of the main drawbacks of antibody-directed enzyme prodrug therapy. Methods: N-terminal and N-, C-terminal fusion CPG2, CNGRC-CPG2, and CNGRC-CPG2-CNGRC, respectively, were PEGylated using polyethylene glycol (PEG) maleimide (40K). We examined the effect of PEGylation on the therapeutic efficacy of the new products. The resulting PEGylated fusion proteins were tested for their stability, ex vivo immunotoxicity, binding capacity to their target on high HT1080, and low A549 APN-expressing cells. The catalytic activity of the resulting PEGylated fusion CPG2 proteins was investigated. Pro-drug "ZD2767P" cytotoxic effect in association with PEG CPG2-CNGRC fusion proteins on cancer cells was studied. Results: Our work demonstrated that the properties of the PEGylated single-fused proteins were significantly improved over that of un-PEGylated fused CPG2, and its kinetic activity and APN-binding affinity were not negatively affected by the PEGylation. Significantly, The PEGylated single-fused CPG2 had lower immunogenicity than the un-PEGylated CPG2. Our results, however, were different in the case of the PEGylated double-fused CPG2. Although its stability in human serum under physiological conditions was not significantly affected, the kinetic activity and its binding affinity to their cellular marker (APN) were substantially reduced. When the study was performed with high and low APN-expressing cancer cell lines, using the prodrug ZD2767p, the PEGylated fusion CPG2 demonstrated cancer cell killing effects. Conclusion: We have successfully produced PEGylated-CNGRC-CPG2, which is bioactive and with lower immunogenicity in ligand-directed enzyme prodrug therapy for cancer treatment.

Structure and mechanism of the γ-glutamyl-γ-aminobutyrate hydrolase SpuA from Pseudomonas aeruginosa.

Polyamines are important regulators in all living organisms and are implicated in essential biological processes including cell growth, differentiation and apoptosis. Pseudomonas aeruginosa possesses an spuABCDEFGHI gene cluster that is involved in the metabolism and uptake of two polyamines: spermidine and putrescine. In the proposed γ-glutamylation-putrescine metabolism pathway, SpuA hydrolyzes γ-glutamyl-γ-aminobutyrate (γ-Glu-GABA) to glutamate and γ-aminobutyric acid (GABA). In this study, crystal structures of P. aeruginosa SpuA are reported, confirming it to be a member of the class I glutamine amidotransferase (GAT) family. Activity and substrate-binding assays confirm that SpuA exhibits a preference for γ-Glu-GABA as a substrate. Structures of an inactive H221N mutant were determined with bound glutamate thioester intermediate or glutamate product, thus delineating the active site and substrate-binding pocket and elucidating the catalytic mechanism. The crystal structure of another bacterial member of the class I GAT family from Mycolicibacterium smegmatis (MsGATase) in complex with glutamine was determined for comparison and reveals a binding site for glutamine. Activity assays confirm that MsGATase has activity for glutamine as a substrate but not for γ-Glu-GABA. The work reported here provides a starting point for further investigation of polyamine metabolism in P. aeruginosa.

Characterization of a Stable Form of Carboxypeptidase G2 (Glucarpidase), a Potential Biobetter Variant, From Acinetobacter sp. 263903-1.

Carboxypeptidase G2 (CPG2) is a bacterial enzyme widely used to detoxify methotrexate (MTX) and in enzyme/prodrug therapy for cancer treatment. However, several drawbacks, such as instability, have limited its efficiency. Herein, we have evaluated the properties of a putative CPG2 from Acinetobacter sp. 263903-1 (AcCPG2). AcCPG2 is compared with a CPG2 derived from Pseudomonas sp. strain RS-16 (PsCPG2), available as an FDA-approved medication called glucarpidase. After modeling AcCPG2 using the I-TASSER program, the refined model was validated by PROCHECK, VERIFY 3D and according to the Z score of the model. Using computational analyses, AcCPG2 displayed higher thermodynamic stability and a lower aggregation propensity than PsCPG2. AcCPG2 showed an optimum pH of 7.5 against MTX and was stable over a pH range of 5-10. AcCPG2 exhibited optimum activity at 50 °C and higher thermal stability at a temperature range of 20-70 °C compared to PsCPG2. The Km value of the purified AcCPG2 toward folate and MTX was 31.36 µM and 44.99 µM, respectively. The Vmax value of AcCPG2 for folate and MTX was 125.80 µmol/min/mg and 48.90  µmol/min/mg, respectively. Accordingly, thermostability and pH versatility makes AcCPG2 a potential biobetter variant for therapeutic applications.

Enhanced Solubility and One-Step Purification of Functional Dimeric Carboxypeptidase G2.

Carboxypeptidase G2 is a bacterial enzyme that catalyzes methotrexate conversion to its inactive forms which are then eliminated via a non-renal pathway in patients with renal disorders during a high-dose methotrexate administration. Due to the increasing demand of this enzyme, it was of interest to simplify its production process. For this reason, we developed a method for production and one-step purification of this enzyme using an intein-mediated system with a chitin-binding affinity tag. The carboxypeptidase G2 gene from Pseudomonas RS16 was optimized, synthesized, cloned into the pTXB1 expression vector and finally transformed into Escherichia coli BL21 (DE3) cells. The optimal condition for the enzyme soluble expression was achieved in 2×YT medium containing 1% glucose at 25°C for 30 h with 0.5 mM IPTG. The enzyme without intein was expressed as inclusion bodies indicating the importance of intein for the protein solubility. The expressed homodimer protein was purified to homogeneity on a chitin affinity column. The Km and kcat values of 6.5 µM and 4.57 s-1, respectively, were obtained for the purified enzyme. Gel filtration analysis indicated that the resulting recombinant protein was a dimer of 83 kDa. Fluorescence and circular dichroism spectroscopy confirmed the enzyme tertiary and secondary structures, respectively. The use of intein-mediated system provided the possibility of the one-step carboxypeptidase G2 purification, paving the way to the application of this enzyme in pharmaceutics.

Ultrasound Enhances ZD2767P-Carboxypeptidase G2 against Chemoresistant Ovarian Cancer Cells by Altering the Intracellular Pharmacokinetics of ZD2767D.

Prodrug-carboxypeptidase G2 (e.g., ZD2767P+CPG2) can realize a targeted treatment where the specific advantage is a lack of CPG2 analogues in humans, but it is limited by low efficacy. Here ultrasound was employed to enhance ZD2767P+CPG2 (i.e., ZD2767P+CPG2+US) against chemoresistant human ovarian cancer cells. The release dynamics of ZD2767D (activated drug) by CPG2 were investigated. The in vitro efficacy was explored in SKOV3 and SKOV3/DDP (cisplatin-resistant subline) cells; spectrophotometry was established to quantify ZD2767P and ZD2767D, and then intracellular pharmacokinetics were evaluated. The in vivo efficacy was validated in both subcutaneous and orthotopic tumors. With insonation, the ZD2767D concentration was increased during an early period. Insonation synergized ZD2767P+CPG2 to enhance cell death and apoptosis, and efficacies in SKOV3 and SKOV3/DDP cells were similar. Intracellular pharmacokinetics of ZD2767D were nonproportional, and insonation increased the peak level, area under the level vs time curve, and mean residence time. In subcutaneous xenografts, ZD2767P+CPG2 and ZD2767P+CPG2+US resulted in volume-inhibitory rates of 20.4% and 26.5% in SKOV3 tumors and 36.8% and 81.6% in SKOV3/DDP tumors, respectively. In the orthotopic tumor model, the survival time in group ZD2767P+CPG2 or ZD2767P+CPG2+US was prolonged compared with group control, in SKOV3 (33.0 ± 3.5 or 39.2 ± 1.8 vs 25.0 ± 1.6 days, p < 0.0001) and SKOV3/DDP (16.2 ± 4.8 or 22.3 ± 7.3 vs 8.7 ± 3.9 days, p = 0.0015) tumors. These data indicated that ZD2767P+CPG2+US was effective against resistant ovarian cancer cells.

Determination of Total Folates in Complex Nutritional Drinks and Supplements Using a Tri-Enzyme Microbiological Method and Michaelis-Menten Kinetics.

Background: Recent development of LC methods for the determination of total folates (vitamin B9) in complex matrixes have been hindered by vitamer interconversion and yield variability. The official microbiological method (AOAC Official Methods of Analysis 944.12 and 960.46) uses an end point turbidity reading to determine folate concentration. However, when measuring complex matrixes, shifts are observed in the growth curves of the microorganism and inaccuracies are introduced to this quantification method. Objective/Methods: In addition to the tri-enzyme digestion of the standard microbiological method, we have applied enzyme modeling of the initial velocity of bacterial growth using Michaelis-Menten kinetics to achieve more accurate and reproducible determinations of total folates. Results/Conclusions: Accuracy determined through spike recovery in Infant/Adult Nutritional Drink and a complex vitamin matrix gave values acceptable to AOAC standards of 85-110%. Repeatability of the low mass fraction analyte measured at micrograms per 100 g yielded relative standard deviations <15% for all matrixes tested, including three standard reference materials.

Production of "biobetter" glucarpidase variants to improve drug detoxification and antibody directed enzyme prodrug therapy for cancer treatment.

Recombinant glucarpidase (formerly: Carboxypeptidase G2, CPG2) is used in Antibody Directed Enzyme Prodrug Therapy (ADEPT) for the treatment of cancer. In common with many protein therapeutics, glucarpidase has a relatively short half-life in serum and, due to the need for the repeated cycles of the ADEPT, its bioavailability may be further diminished by neutralizing antibodies produced by patients. PEGylation and fusion with human serum albumin (HSA) are two approaches that are commonly employed to increase the residency time of protein therapeutics in blood, and also to increase the half-lives of the proteins in vivo. To address this stability and the immunogenicity problems, 'biobetter' glucarpidase variants, mono-PEGylated glucarpidase, and HSA fused glucarpidase by genetic fusion with albumin, were produced. Biochemical and bioactivity analyses, including anti-proliferation, bioassays, circular dichroism, and in vitro stability using human blood serum and immunoassays, demonstrated that the functional activities of the designed glucarpidase conjugates were maintained. The immunotoxicity studies indicated that the PEGylated glucarpidase did not significantly induce T-cell proliferation, suggesting that glucarpidase epitopes were masked by the PEG moiety. However, free glucarpidase and HSA-glucarpidase significantly increased T-cell proliferation compared with the negative control. In the latter case, this might be due to the type of expression system used or due to trace impurities associated with the highly purified (99.99%) recombinant HSA-glucarpidase. Both PEGylated glucarpidase and HAS-glucarpidase exhibit more stability in human serum and were more resistant to key human proteases relative to native glucarpidase. To our knowledge, this study is the first to report stable and less immunogenic glucarpidase variants produced by PEGylation and fusion with HSA. The results suggest that they may have better efficacy in drug detoxification and ADEPT, thereby improving this cancer treatment strategy.

Characterisation of the Carboxypeptidase G2 Catalytic Site and Design of New Inhibitors for Cancer Therapy.

The enzyme carboxypeptidase G2 (CPG2) is used in antibody-directed enzyme prodrug therapy (ADEPT) to catalyse the formation of an active drug from an inert prodrug. Free CPG2 in the bloodstream must be inhibited before administration of the prodrug in order to avoid a systemic reaction in the patient. Although a few small-molecule CPG2 inhibitors have been reported, none has been taken forward thus far. This lack of progress is due in part to a lack of structural understanding of the CPG2 active site as well as the absence of small molecules that can block the active site whilst targeting the complex for clearance. The work described here aimed to address both areas. We report the structural/functional impact of extensive point mutation across the putative CPG2 catalytic site and adjacent regions for the first time, revealing that residues outside the catalytic region (K208A, S210A and T357A) are crucial to enzyme activity. We also describe novel molecules that inhibit CPG2 whilst maintaining the accessibility of galactosylated moieties aimed at targeting the enzyme for clearance. This work acts as a platform for the future development of high-affinity CPG2 inhibitors that occupy new chemical space and will advance the safe application of ADEPT in cancer treatment.

Isolation and molecular characterization of novel glucarpidases: Enzymes to improve the antibody directed enzyme pro-drug therapy for cancer treatment.

Repeated cycles of antibody-directed enzyme pro-drug therapy (ADEPT) and the use of glucarpidase in the detoxification of cytotoxic methotrexate (MTX) are highly desirable during cancer therapy but are hampered by the induced human antibody response to glucarpidase. Novel variants of glucarpidase (formal name: carboxypeptidase G2, CPG2) with epitopes not recognized by the immune system are likely to allow repeated cycles of ADEPT for effective cancer therapy. Towards this aim, over two thousand soil samples were collected and screened for folate hydrolyzing bacteria using folate as the sole carbon source. The work led to the isolation and the characterization of three new glucarpidase producing strains, which were designated as: Pseudomonas lubricans strain SF168, Stenotrophomonas sp SA and Xenophilus azovorans SN213. The CPG2 genes of Xenophilus azovorans SN213 (named Xen CPG2) and Stenotrophomonas sp SA (named Sten CPG2) were cloned and molecularly characterized. Both Xen CPG2 and Sten CPG2 share very close amino acid sequences (99%); we therefore, focused on the study of Xen CPG2. Finally, we demonstrated that a polyclonal antibody raised against our new CPG2, Xen CPG2, does not react with the CPG2 from Pseudomonas sp. strain RS-16 (Ps CPG2) that are currently in clinical use. The two enzymes, therefore could potentially be used consecutively in the ADEPT protocol to minimize the effect of the human antibody response that hampers current treatment with Ps CPG2. The identified novel CPG2 in this study will, therefore, pave the way for safer antibody directed enzyme pro-drug therapy for cancer treatment.

Biphasically Modulating the Activity of Carboxypeptidase G2 with Ultrasound.

BACKGROUND/AIMS: Carboxypeptidase G2 (CPG2) has been used for cancer prodrug therapy to realize the targeted release of active drugs, but there yet lacks a means to modulate the CPG2 activity. Here ultrasound was used to modulate the CPG2 activity. METHODS: The activity of insonated CPG2 was determined, and then underlying biochemical (i.e., monomer, dimer and conformation) and ultrasonic (i.e., heat and cavitation) mechanisms were explored. RESULTS: Ultrasound (1.0 MHz) increased or decreased the enzymatic activity; the activity decreased as zero- or first-order kinetics, depending on the intensity. L1 (10 W/cm2 for 200 s) improved the activity via increasing the specific activity. L2 or L3 (20 W/cm2 for 1200 or 3000 s) decreased the activity via disassembling the dimer, degrading the monomer, inducing glycosylation, transforming conformation and decreasing the specific activity. An increase or a slight decrease of activity attributable to 10 W/cm2 was reversible, but the activity decrease due to 20 W/cm2 was irreversible. The enzymatic modulation was realized via cavitation. CONCLUSION: Ultrasound can biphasically modulate the CPG2 activity, and can be employed in the CPG2-prodrug therapy to adjust the release and moles of active drugs.

Engineering carboxypeptidase G2 circular permutations for the design of an autoinhibited enzyme.

Carboxypeptidase G2 (CPG2) is an Food and Drug Administration (FDA)-approved enzyme drug used to treat methotrexate (MTX) toxicity in cancer patients receiving MTX treatment. It has also been used in directed enzyme-prodrug chemotherapy, but this strategy has been hampered by off-site activation of the prodrug by the circulating enzyme. The development of a tumor protease activatable CPG2, which could be achieved using a circular permutation of CPG2 fused to an inactivating 'prodomain', would aid in these applications. We report the development of a protease accessibility-based screen to identify candidate sites for circular permutation in proximity of the CPG2 active site. The resulting six circular permutants showed similar expression, structure, thermal stability, and, in four cases, activity levels compared to the wild-type enzyme. We rationalize these results based on structural models of the permutants obtained using the Rosetta software. We developed a cell growth-based selection system, and demonstrated that when fused to periplasm-directing signal peptides, one of our circular permutants confers MTX resistance in Escherichia coli with equal efficiency as the wild-type enzyme. As the permutants have similar properties to wild-type CPG2, these enzymes are promising starting points for the development of autoinhibited, protease-activatable zymogen forms of CPG2 for use in therapeutic contexts.

Soluble expression, purification and functional characterisation of carboxypeptidase G2 and its individual domains.

Due to its applications in the treatment of cancer and autoimmune diseases, the 42 kDa zinc-dependent metalloenzyme carboxypeptidase G2 (CPG2) is of great therapeutic interest. An X-ray crystal structure of unliganded CPG2 reported in 1997 revealed the domain architecture and informed early rational drug design efforts, however further efforts at co-crystallization of CPG2 with ligands, substrates or inhibitors have not been reported. Thus key features of CPG2 such as the location of the active site, the presence of additional ligand-binding sites, stability, oligomeric state, and the molecular basis of activity remain largely unknown, with the current working understanding of CPG2 activity based primarily on computational modelling. To facilitate renewed efforts in CPG2 structural biology, we report the first high-yield (250 mg L(-1)) recombinant expression (and purification) of soluble and active CPG2 using the Escherichia coli expression system. We used this protocol to produce full-length enzyme, as well as protein fragments corresponding to the individual catalytic and dimerization domains, and the activity and stability of each construct was characterised. We adapted our protocol to allow for uniform incorporation of NMR labels ((13)C, (15)N and (2)H) and present preliminary solution-state NMR spectra of high quality. Taken together, our results offer a route for production and solution-state characterization that supports renewed effort in CPG2 structural biology as well as design of significantly truncated CPG2 proteins, which retain activity while yielding (potentially) improved immunogenicity.

The 46 kDa dimeric protein from Variovorax paradoxus shows faster methotrexate degrading activity in its nanoform compare to the native enzyme.

Methotrexate degrading enzymes are required to overcome the toxicity of the methotrexate while treating the cancer. The enzyme from Variovorax paradoxus converts the methotrexate in to non toxic products. Methotrexate degrading enzyme from V. paradoxus is a dimeric protein with a molecular mass of 46 kDa and it acts on casein and gelatin. This enzyme is optimally active at pH 7.5 and 40°C and nanoparticles of this enzyme were prepared by desolvation-crosslinking method. Enzyme nanoparticles could degrade methotrexate faster than the native enzyme and they show lower Km compare to the native enzyme. Enzyme nanoparticles show better thermostability and they were stable for much longer time in the serum compare to the native enzyme. Enzyme nanoparticles show better functionality than the native enzyme while clearing the methotrexate added to the serum suggesting their advantage over the native enzyme for the therapeutic and biotechnological applications.

Nanosecond electric pulses deprive zinc ions of carboxypeptidase G2.

Nanosecond electric pulses (nsEP, 10kV/cm with a pulse duration of 8, 16 or 24ns) inhibited the activity of carboxypeptidase G2 (CPG2), a zinc-dependent homodimer; the relative activity was <20% when the total exposure time was >120s. No alterations were detected in electrophoresis, chromatography, mass spectroscopy and circular dichroism, thus demonstrating intactness of the apoenzyme. Inductively coupled plasma-mass spectrometry indicated that zinc levels were 3.30µg/mg protein in control CPG2, and decreased to 0.40, 0.12 or 0.38µg/mg protein after 240s of 8-, 16- or 24-ns pulses, respectively. In CPG2 exposed to 240s of 8-, 16- and 24-ns pulses, the reloading of zinc with redialysis recovered the activity to 94.7±3.4%, 84.0±5.2% and 81.7±7.0%, respectively (p=0.0853, 0.0741, 0.0668). These data demonstrated that nsEP inhibited CPG2 via removal of zinc, and that nsEP can be used to modulate CPG2.

Structural insights into the hydrolysis and polymorphism of methotrexate polyglutamate by zebrafish γ-glutamyl hydrolase.

γ-Glutamyl hydrolases (γGH) catalyze the hydrolysis of γ-linked glutamate residues from the polyglutamyl of folates and antifolates, such as methotrexate (MTX), a widely used anticancer drug. We describe the first crystal structures of the endopeptidase-type γGH (zγGH) from zebrafish and the mutant complexes with MTX(Glu)5 and hydrolyzed MTX(Glu)1, revealing the complete set of key residues involved in hydrolysis as well as the substrate-binding subsites (-1 to +2). The side chain of Phe20 and the 6-methylpterin ring of MTX(Glu)5 invoke π-π interactions to promote distinct concerted conformational alterations involving ∼90° rotations in the complexes with the zγGH-C108A and zγGH-H218N mutant proteins. The structural geometries of the MTX(Glu)5 and hydrolyzed MTX(Glu)1 in the mutant complexes differ significantly from those of the previously known MTX(Glu)1, providing polymorphic information. Together with the structural comparison and the activity analysis, these results shed light on the catalytic mechanism and substrate recognition of zγGH and other γ-glutamyl hydrolases.

Molecular modeling approach to predict a binding mode for the complex methotrexate-carboxypeptidase G2.

Carboxypeptidase G(2) (CPG(2)) is a zinc-metalloenzyme employed in a range of cancer chemotherapy strategies by activating selectively nontoxic prodrugs into cytotoxic drugs in tumor as well as in the treatment of intoxication caused by high-doses of the anticancer drug methotrexate (MTX). CPG(2) catalyzes the hydrolytic cleavage of C-terminal of glutamate moiety from folic acid and analogues. Regardless of its extensive application, its mechanism of catalysis has not yet been determined and, so far, no co-crystallized complex has been published. So, in this study, molecular docking and a short molecular dynamics (MD) simulation sampling scheme, as a function of temperature, were performed to investigate a possible binding mode for MTX, a recognized substrate of CPG(2). The findings suggested that MTX interacts possibly in quite specific points of the CPG(2) active site, which are probably responsible for the molecular recognition and cleavage procedures. The MTX substrate fits well in the catalytic site by accommodating the pteridine moiety in an adjacent pocket to the active site whereas a glutamate moiety is pointed toward the protein surface. Additionally, a glutamate residue can interact with a crystallization water molecule in the active site, supporting its activation as a nucleophilic group.

Biochemical and structural properties of gamma-glutamyl transpeptidase from Geobacillus thermodenitrificans: an enzyme specialized in hydrolase activity.

Gamma-glutamyltranspeptidases (gamma-GTs) catalyze the transfer of the gamma-glutamyl moiety of glutathione and related gamma-glutamyl amides to water (hydrolysis) or to amino acids and peptides (transpeptidation) and play a key role in glutathione metabolism. Recently, gamma-GTs have been considered attractive pharmaceutical targets for cancer and useful tools to produce gamma-glutamyl compounds. To find out gamma-GTs with special properties we have chosen microorganisms belonging to Geobacillus species which are source of several thermostable enzymes of potential interest for biotechnology. gamma-GT from Geobacillus thermodenitrificans (GthGT) was cloned, expressed in Escherichia coli, purified to homogeneity and characterized. The enzyme, synthesized as a precursor homotetrameric protein of 61-kDa per subunit, undergoes an internal post-translational cleavage of the 61 kDa monomer into 40- and 21-kDa shorter subunits, which are then assembled into an active heterotetramer composed of two 40- and two 21-kDa subunits. The kinetic characterization of the hydrolysis reaction using L-glutamic acid gamma-(4-nitroanilide) as the substrate reveals that the active enzyme has K(m) 7.6 microM and V(max) 0.36 micromol min/mg. The optimum pH and temperature for the hydrolysis activity are 7.8 and 52 degrees C, respectively. GthGT hydrolyses the physiological antioxidant glutathione, suggesting an involvement of the enzyme in the cellular defense mechanism against oxidative stress. Unlike other gamma-GTs, the mutation of the highly conserved catalytic nucleophile, Thr353, abolishes the post-translational cleavage of the pro-enzyme, but does not completely block the hydrolytic action. Furthermore, GthGT does not show any transpeptidase activity, suggesting that the enzyme is a specialized gamma-glutamyl hydrolase. The GthGT homology-model structure reveals peculiar structural features, which should be responsible for the different functional properties of the enzyme and suggests the structural bases of protein thermostability.

Hyperpolarized (13)C magnetic resonance detection of carboxypeptidase G2 activity.

Carboxypeptidase G2 (CPG2) is a bacterial enzyme that is currently employed in a range of targeted cancer chemotherapy strategies such as gene-directed enzyme prodrug therapy (GDEPT). Employing dynamic nuclear polarization (DNP) and natural abundance (13)C magnetic resonance spectroscopy (MRS), we observed the CPG2-mediated conversion of a novel hyperpolarized reporter probe 3,5-difluorobenzoyl-L-glutamic acid (3,5-DFBGlu) to 3,5-difluorobenzoic acid (3,5-DFBA) and L-glutamic acid (L-Glu) in vitro. Isotopic labeling of the relevant nuclei with (13)C in 3,5-DFBGlu or related substrates will yield a further factor of 100 increase in the signal-to-noise. We discuss the feasibility of translating these experiments to generate metabolic images of CPG2 activity in vivo.

Recombinant zebrafish {gamma}-glutamyl hydrolase exhibits properties and catalytic activities comparable with those of mammalian enzyme.

A cDNA encoding for zebrafish gamma-glutamyl hydrolase (gammaGH) was cloned and inserted into a pET43.1a vector via SmaI and EcoRI sites and expressed in Rosetta (DE3) cells as a Nus-His-tag fusion enzyme (NH-zgammaGH). After induction with isopropyl thiogalactoside, the enzyme was purified with a Ni-Sepharose column, and approximately 8 mg of pure enzyme was obtained per liter of culture. The primary sequence of the recombinant zgammaGH was similar to mammalian gammaGH. Thrombin digestion of this NH-zgammaGH fusion protein resulted in zgammaGH with approximately 2-fold higher catalytic activity compared with the NH-zgammaGH fusion enzyme. This recombinant zgammaGH is active and exhibits comparable endopeptidase activity with folate substrate and antifolate drug methotrexate. Use of this recombinant zgammaGH significantly increased efficiency in folylpolyglutamate hydrolysis for folate analysis compared with current protocols.