Strain RS-16 Research Articles

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 31.36µM and 44.99µM, respectively. The Vmax value of AcCPG2 for folate and MTX was 125.80µmol/min/mg and 48.90 µmol/min/mg, respectively. Accordingly, thermostability and pH versatility makes AcCPG2 a potential biobetter variant for therapeutic applications.

Production of “biobetter” variants of glucarpidase with enhanced enzyme activity

Glucarpidase, also known as carboxypeptidase G2, is a Food and Drug Administration-approved enzyme used in targeted cancer strategies such as antibody-directed enzyme prodrug therapy (ADEPT). It is also used in drug detoxification when cancer patients have excessive levels of the anti-cancer agent methotrexate. The application of glucarpidase is limited by its potential immunogenicity and limited catalytic efficiency. To overcome these pitfalls, mutagenesis was applied to the glucarpidase gene of Pseudomonas sp. strain RS-16 to isolate three novels “biobetter” variants with higher specific enzyme activity. DNA sequence analysis of the genes for the variants showed that each had a single point mutation, resulting in the amino acid substitutions: I100 T, G123S and T239 A. Km, Vmax and Kcat measurements confirmed that each variant had increased catalytic efficiency relative to wild type glucarpidase.Additionally, circular dichroism studies indicated that they had a higher alpha-helical content relative to the wild type enzyme. However, three different software packages predicted that they had reduced protein stability, which is consistent with having higher activities as a tradeoff. The novel glucarpidase variants presented in this work could pave the way for more efficient drug detoxification and might allow dose escalation during chemotherapy. They also have the potential to increase the efficiency of ADEPT and to reduce the number of treatment cycles, thereby reducing the risk that patients will develop antibodies to glucarpidase.

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.

Functional Redundancy of Linuron Degradation in Microbial Communities in Agricultural Soil and Biopurification Systems.

The abundance of libA, encoding a hydrolase that initiates linuron degradation in the linuron-metabolizing Variovorax sp. strain SRS16, was previously found to correlate well with linuron mineralization, but not in all tested environments. Recently, an alternative linuron hydrolase, HylA, was identified in Variovorax sp. strain WDL1, a strain that initiates linuron degradation in a linuron-mineralizing commensal bacterial consortium. The discovery of alternative linuron hydrolases poses questions about the respective contribution and competitive character of hylA- and libA-carrying bacteria as well as the role of linuron-mineralizing consortia versus single strains in linuron-exposed settings. Therefore, dynamics of hylA as well as dcaQ as a marker for downstream catabolic functions involved in linuron mineralization, in response to linuron treatment in agricultural soil and on-farm biopurification systems (BPS), were compared with previously reported libA dynamics. The results suggest that (i) organisms containing either libA or hylA contribute simultaneously to linuron biodegradation in the same environment, albeit to various extents, (ii) environmental linuron mineralization depends on multispecies bacterial food webs, and (iii) initiation of linuron mineralization can be governed by currently unidentified enzymes. A limited set of different isofunctional catabolic gene functions is known for the bacterial degradation of the phenylurea herbicide linuron, but the role of this redundancy in linuron degradation in environmental settings is not known. In this study, the simultaneous involvement of bacteria carrying one of two isofunctional linuron hydrolysis genes in the degradation of linuron was shown in agricultural soil and on-farm biopurification systems, as was the involvement of other bacterial populations that mineralize the downstream metabolites of linuron hydrolysis. This study illustrates the importance of the synergistic metabolism of pesticides in environmental settings.

Dynamics of the Linuron HydrolaselibAGene Pool Size in Response to Linuron Application and Environmental Perturbations in Agricultural Soil and On-Farm Biopurification Systems

libA, a gene encoding a novel type of linuron hydrolase, was recently identified in the linuron-mineralizing Variovorax sp. strain SRS16. In order to assess the contribution of libA to linuron degradation in environmental settings, libA abundance was monitored in response to the application of linuron and to environmental perturbations in agricultural soil microcosms and microcosms simulating the matrix of on-farm biopurification systems. libA numbers were measured by real-time PCR and linked to reported data of Variovorax community composition and linuron mineralization capacity. In the soil microcosms and one biopurification system setup, libA numbers responded to the application of linuron and environmental changes in congruency with the modulation of linuron mineralization capacity and the occurrence of a particular Variovorax phylotype (phylotype A). However, in another biopurification system setup, no such correlations were found. Our data suggest that in the simulated environmental settings, the occurrence of libA can be linked to the linuron mineralization capacity and that libA is primarily hosted by Variovorax phylotype A strains. However, the results also suggest that, apart from libA, other, as-yet-unknown isofunctional genes play an important role in linuron mineralization in the environment.

A Novel Hydrolase Identified by Genomic-Proteomic Analysis of Phenylurea Herbicide Mineralization by Variovorax sp. Strain SRS16

The soil bacterial isolate Variovorax sp. strain SRS16 mineralizes the phenylurea herbicide linuron. The proposed pathway initiates with hydrolysis of linuron to 3,4-dichloroaniline (DCA) and N,O-dimethylhydroxylamine, followed by conversion of DCA to Krebs cycle intermediates. Differential proteomic analysis showed a linuron-dependent upregulation of several enzymes that fit into this pathway, including an amidase (LibA), a multicomponent chloroaniline dioxygenase, and enzymes associated with a modified chlorocatechol ortho-cleavage pathway. Purified LibA is a monomeric linuron hydrolase of ∼55 kDa with a K(m) and a V(max) for linuron of 5.8 μM and 0.16 nmol min⁻¹, respectively. This novel member of the amidase signature family is unrelated to phenylurea-hydrolyzing enzymes from Gram-positive bacteria and lacks activity toward other tested phenylurea herbicides. Orthologues of libA are present in all other tested linuron-degrading Variovorax strains with the exception of Variovorax strains WDL1 and PBS-H4, suggesting divergent evolution of the linuron catabolic pathway in different Variovorax strains. The organization of the linuron degradation genes identified in the draft SRS16 genome sequence indicates that gene patchwork assembly is at the origin of the pathway. Transcription analysis suggests that a catabolic intermediate, rather than linuron itself, acts as effector in activation of the pathway. Our study provides the first report on the genetic organization of a bacterial pathway for complete mineralization of a phenylurea herbicide and the first report on a linuron hydrolase in Gram-negative bacteria.

Enhancement of growth and salt tolerance of red pepper seedlings (Capsicum annuum L.) by regulating stress ethylene synthesis with halotolerant bacteria containing 1-aminocyclopropane-1-carboxylic acid deaminase activity

Three 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing halotolerant bacteria were isolated from West Coast soil of Yellow Sea, Incheon, South Korea and evaluated for their efficiency in improving red pepper plant growth under salt stress. The strains RS16, RS656 and RS111 were identified by 16S rRNA gene sequencing as Brevibacterium iodinum, Bacillus licheniformis and Zhihengliuela alba, respectively. Two hour exposure of 100, 150 and 200 mM NaCl stress on 8 day old red pepper seedlings caused 44, 64 and 74% increase ethylene production, while at 150 mM NaCl stress, inoculation of B. licheniformis RS656, Z. alba RS111, and Br. iodinum RS16 reduces ethylene production by 44, 53 and 57%, respectively. Similarly, 3 week old red pepper plants were subjected to salt stress for two weeks and approximately ∼50% reduction in growth recorded at 150 mM NaCl stress compared to negative control whereas bacteria inoculation significantly increase the growth compared to positive control. Salt stress also caused 1.3-fold reduction in the root/shoot dry weight ratio compared to the absence of salt while bacteria inoculation retained the biomass allocation similar to control plants. The salt tolerance index (ratio of biomass of salt stressed to non-stressed plant) was also significantly increased in inoculated plants compared to non-inoculated. Increase nutrient uptakes under salt stress by red pepper further evident that bacteria inoculation ameliorates salt stress effect. In summary, this study indicates that the use of ACC deaminase-producing halotolerant bacteria mitigates the salt stress by reducing salt stress-induced ethylene production on growth of red pepper plants.

Functional Overexpression and Purification of a Codon Optimized Synthetic Glucarpidase (Carboxypeptidase G2) in Escherichia coli

Glucarpidase (former name: carboxypeptidase G2, or CPG2) is a bacterial enzyme that is widely used in detoxification of the cytotoxic drug, methotrexate, and in Antibody Directed Enzyme Prodrug Therapy for cancer treatment. The glucarpidase gene of Pseudomonas sp. strain RS-16 was previously cloned in E coli, but expresses at a level that is approximately 100-fold lower than in the native strain. In this study, a synthetic gene coding for glucarpidase was codon-optimised and synthesized for maximum expression in E. coli using the vector pET28a. Our work indicated that the enzyme was expressed to ~60% of the total host protein and that purification of the recombinant His-tagged protein could be achieved in a single step by Ni(2+) charged column chromatography. The synthetic recombinant glucarpidase expressed within this system was biologically active and zinc dependant. Our study showed that Mg(2+) as well as Mn(2+) ions inhibit the activity of the recombinant enzyme.

Constitutive mineralization of low concentrations of the herbicide linuron by aVariovoraxsp. strain

The mineralization of the herbicide linuron at concentrations of microg and mg L(-1) was studied in liquid batch experiments with Variovorax sp. strain SRS16. The strain was highly efficient at mineralizing a range of linuron concentrations (0.002-10 mg L(-1)) with 20-60% of the added (14)C-ring-labeled linuron metabolized to (14)CO(2) within hours to days depending on the initial linuron concentration and incubation period. At mg L(-1) linuron concentrations the mineralization activity by SRS16 was inducible and a shift to constitutive mineralization activity was apparent with a reduction in the linuron concentration to microg L(-1) levels. This study revealed that strain SRS16 is a promising candidate for bioaugmentation of water or soil resources contaminated with low linuron concentrations.

The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae contains two active-site histidine residues

The catalytic and structural properties of the H67A and H349A dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. On the basis of sequence alignment with the carboxypeptidase from Pseudomonas sp. strain RS-16, both H67 and H349 were predicted to be Zn(II) ligands. The H67A DapE enzyme exhibited a decreased catalytic efficiency (180-fold) compared with wild-type (WT) DapE towards N-succinyldiaminopimelic acid. No catalytic activity was observed for H349A under the experimental conditions used. The electronic paramagnetic resonance (EPR) and electronic absorption data indicate that the Co(II) ion bound to H349A-DapE is analogous to that of WT DapE after the addition of a single Co(II) ion. The addition of 1 equiv of Co(II) to H67A DapE provides spectra that are very different from those of the first Co(II) binding site of the WT enzyme, but that are similar to those of the second binding site. The EPR and electronic absorption data, in conjunction with the kinetic data, are consistent with the assignment of H67 and H349 as active-site metal ligands for the DapE from H. influenzae. Furthermore, the data suggest that H67 is a ligand in the first metal binding site, while H349 resides in the second metal binding site. A three-dimensional homology structure of the DapE from H. influenzae was generated using the X-ray crystal structure of the DapE from Neisseria meningitidis as a template and superimposed on the structure of the aminopeptidase from Aeromonas proteolytica (AAP). This homology structure confirms the assignment of H67 and H349 as active-site ligands. The superimposition of the homology model of DapE with the dizinc(II) structure of AAP indicates that within 4.0 A of the Zn(II) binding sites of AAP all of the amino acid residues of DapE are nearly identical.

Promicromonospora kroppenstedtii sp. nov., isolated from sandy soil

A new actinobacterial strain, RS16(T), was isolated from sandy soil collected in Zamora, Spain, and was studied to determine its taxonomic position. A neighbour-joining tree based on 16S rRNA gene sequence analysis revealed that the novel isolate formed an independent branch between Promicromonospora sukumoe DSM 44121(T) and Promicromonospora citrea DSM 43110(T). Levels of 16S rRNA gene sequence similarity between the novel isolate and its phylogenetic neighbours ranged from 98.7 to 98.9 %. Chemotaxonomic properties, such as the predominant menaquinone and polar lipids, supported the assignment of the novel isolate to the genus Promicromonospora, however, a significant number of physiological differences were found between the novel isolate and the other recognized species of the genus Promicromonospora. On the basis of these results, it is proposed that strain RS16(T) represents a novel species of the genus Promicromonospora, for which the name Promicromonospora kroppenstedtii sp. nov. is proposed. The type strain is RS16(T) (=DSM 19349(T)=LMG 24382(T)).

Rapid Mineralization of the Phenylurea Herbicide Diuron by Variovorax sp. Strain SRS16 in Pure Culture and within a Two-Member Consortium

The phenylurea herbicide diuron [N-(3,4-dichlorophenyl)-N,N-dimethylurea] is widely used in a broad range of herbicide formulations, and consequently, it is frequently detected as a major water contaminant in areas where there is extensive use. We constructed a linuron [N-(3,4-dichlorophenyl)-N-methoxy-N-methylurea]- and diuron-mineralizing two-member consortium by combining the cooperative degradation capacities of the diuron-degrading organism Arthrobacter globiformis strain D47 and the linuron-mineralizing organism Variovorax sp. strain SRS16. Neither of the strains mineralized diuron alone in a mineral medium, but combined, the two strains mineralized 31 to 62% of the added [ring-U-(14)C]diuron to (14)CO(2), depending on the initial diuron concentration and the cultivation conditions. The constructed consortium was used to initiate the degradation and mineralization of diuron in soil without natural attenuation potential. This approach led to the unexpected finding that Variovorax sp. strain SRS16 was able to mineralize diuron in a pure culture when it was supplemented with appropriate growth substrates, making this strain the first known bacterium capable of mineralizing diuron and representatives of both the N,N-dimethyl- and N-methoxy-N-methyl-substituted phenylurea herbicides. The ability of the coculture to mineralize microgram-per-liter levels of diuron was compared to the ability of strain SRS16 alone, which revealed the greater extent of mineralization by the two-member consortium (31 to 33% of the added [ring-U-(14)C]diuron was mineralized to (14)CO(2) when 15.5 to 38.9 mug liter(-1) diuron was used). These results suggest that the consortium consisting of strains SRS16 and D47 could be a promising candidate for remediation of soil and water contaminated with diuron and linuron and their shared metabolite 3,4-dichloroaniline.

ArgE-Encoded N-Acetyl-l-Ornithine Deacetylase from Escherichia coli Contains a Dinuclear Metalloactive Site

The catalytic and structural properties of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were investigated. On the basis of kinetic and ITC (isothermal titration calorimetry) data, Zn(II) binds to ArgE with Kd values that differ by approximately 20 times. Moreover, ArgE exhibits approximately 90% of its full catalytic activity upon addition of one metal ion. Therefore, ArgE behaves similarly to the aminopeptidase from Aeromonas proteolytica (AAP) in that one metal ion is the catalytic metal ion while the second likely plays a structural role. The N-acetyl-L-ornithine (NAO) deacetylase activity of ArgE showed a linear temperature dependence from 20 to 45 degrees C, indicating that the rate-limiting step does not change over this temperature range. The activation energy for NAO hydrolysis by ArgE was 25.6 kJ/mol when loaded with Zn(II) and 34.3 kJ/mol when loaded with Co(II). Electronic absorption and EPR (electron paramagnetic resonance) spectra of [Co x (ArgE)] and [CoCo(ArgE)] indicate that both divalent metal binding sites are five coordinate. In addition, EPR data show clear evidence of spin-spin coupling between the Co(II) ions in the active site but only after addition of a second equivalent of Co(II). Combination of these data provides the first physical evidence that the ArgE from E. coli contains a dinuclear Zn(II) active site, similar to AAP and the carboxypeptidase G2 from Pseudomonas sp. strain RS-16 (CPG2).

The Catalytic Role of Glutamate 151 in the Leucine Aminopeptidase from Aeromonas proteolytica

Glutamate 151 has been proposed to act as the general acid/base during the peptide hydrolysis reaction catalyzed by the co-catalytic metallohydrolase from Aeromonas proteolytica (AAP). However, to date, no direct evidence has been reported for the role of Glu-151 during catalytic turnover by AAP. In order to elucidate the catalytic role of Glu-151, altered AAP enzymes have been prepared in which Glu-151 has been substituted with a glutamine, an alanine, and an aspartate. The Michaelis constant (K(m)) does not change upon substitution to aspartate or glutamine, but the rate of the reaction changes drastically in the following order: glutamate (100% activity), aspartate (0.05%), glutamine (0.004%), and alanine (0%). Examination of the pH dependence of the kinetic constants k(cat) and K(m) revealed a change in the pK(a) of a group that ionizes at pH 4.8 in recombinant leucine aminopeptidase (rAAP) to 4.2 for E151D-AAP. The remaining pK(a) values at 5.2, 7.5, and 9.9 do not change. Proton inventory studies indicate that one proton is transferred in the rate-limiting step of the reaction at pH 10.50 for both rAAP and E151D-AAP, but at pH 6.50 two protons and general solvation effects are responsible for the observed effects in the reaction catalyzed by rAAP and E151D-AAP, respectively. Based on these data, Glu-151 is intrinsically involved in the peptide hydrolysis reaction catalyzed by AAP and can be assigned the role of a general acid and base.

A new crystal form of carboxypeptidase G2 from Pseudomonas sp. strain RS-16 which is more amenable to structure determination.

Carboxypeptidase G(2) is a zinc-dependent exopeptidase which has applications in cancer therapy. Crystallization of carboxypeptidase G(2), first achieved more than a decade ago, yields large crystals; however, problems with non-isomorphism between native crystals as well as failure to obtain any useful heavy-atom derivatives have precluded structure solution. A modification of the crystallization protocol leading to a promising new crystal form which diffracts beyond 3.0 A resolution on a rotating-anode source is now reported. These crystals are readily indexed on an apparent C-centred orthorhombic lattice with a = 81.35, b = 230.9 and c = 105.5 A, but the correct crystal system is monoclinic. The crystals have space group P2(1), with a = 81.35, b = 105.5, c = 122.4 A and beta = 109.3 degrees. There are two possible non-equivalent monoclinic indexings with these lattice constants. A partial native data set collected at the SRS, Daresbury, indicates that 1.9 A diffraction is attainable. Structure determination using MIR methods is in progress.

Crystallization and preliminary crystallographic analysis of carboxypeptidase G2 from Pseudomonas sp. strain RS-16

Carboxypeptidase G2, a zinc metalloenzyme isolated from Pseudomonas sp. strain RS-16, which catalyses the hydrolytic cleavage of reduced and non-reduced folates to pteroates and L-glutamate, has been crystallized from polyethylene glycol (average Mr 4000) by vapour diffusion. The crystal symmetry is monoclinic C2, with unit cell dimensions a = 206 A, b = 82 A, c = 116 A and beta = 118 degrees. The molecular mass and volume of the unit cell suggest that there are two dimers of the enzyme in the asymmetric unit. The crystals diffract to at least 3.0 A and are suitable for X-ray structure analysis.

Carboxypeptidase G 2 enhances trimetrexate cytotoxicity in CCRF-cem cell lines sensitive and resistant to methotrexate

Carboxypeptidase G 2 (CPG 2), an enzyme produced by Pseudomonas strain RS-16, degrades folates as well as methotrexate and other folic acid analogs such as aminopterin and dichloromethotrexate, but not the “non-classical” folate antagonist trimetrexate (TMTX). The possibility of enhancing TMTX cytotoxicity of CPG 2 induced depletion of intracellular folates was investigated in human leukemic CCRF-CEM cells and in three methotrexate resistant sublines of these cells with different mechanisms of resistance, CCRF-CEM/E, a subline with increased DHFR; CCRF-CEM/P, a subline defective in polyglutamylation and CCRF-CEM/T, a subline with impaired MTX uptake. The cytotoxic effect was detected using a colorimetric assay with MTT. Dose-effect relationships of single drugs alone and in combination were analyzed by the median effect principle and by the combination indices for the quantitation of synergism or antagonism with the aid of a microcomputer. TMTX alone was very effective on the parent and all the resistant cell lines (CCRF-CEM/E, CCRF-CEM/P, CCRF-CEM/T) with ED 50 values in the nanomolar range (1.4, 1.6, 1.5 and 0.7 n m, respectively) following 5 days of exposure. The ED 50s of CPG 2 for these cell lines were 3.5, 2.6, 26.6 and 7.9 × 10 −5 U/ml, respectively. A synergistic cytotoxic effect of TMTX after simultaneous continuous exposure was observed with CPG 2 on CCRF-CEM cells and on the three resistant cell lines.

The potential of carboxypeptidase G2-antibody conjugates as anti-tumour agents. I. Preparation of antihuman chorionic gonadotrophin-carboxypeptidase G2 and cytotoxicity of the conjugate against JAR choriocarcinoma cells in vitro.

Carboxypeptidase G2, a zinc metalloenzyme isolated from Pseudomonas sp. strain RS-16, which catalyses the hydrolytic cleavage of reduced and non-reduced folates to pteroates and L-glutamate, has been linked to a monoclonal antibody (W14A) raised to human chorionic gonadotrophin. The coupling efficiency and retention of antibody and enzymatic activities are compared for three separate methods of preparing 1:1 conjugates. Preliminary in vitro studies on the cytotoxicity of the free enzyme and the conjugated enzyme towards JAR choriocarcinoma cells are reported. Despite the limitations of the in vitro model, it could be demonstrated that a significant proportion of 10(6) choriocarcinoma cells lost viability when exposed to either free or conjugated enzyme for 72 hours at concentrations of carboxypeptidase G2 of 1-3 units ml-1 of medium.

Purification and properties of carboxypeptidase G2 from Pseudomonas sp. strain RS-16. Use of a novel triazine dye affinity method.

A folate-degrading enzyme, carboxypeptidase G2, has been purified on a large scale from Pseudomonas sp. strain RS-16. Homogeneous enzyme was obtained by a three-step procedure involving ion-exchange chromatography and a novel triazine dye (affinity) chromatography step which utilizes Zn2+ to promote adsorption of the enzyme. Enzyme was selectively eluted by the use of a chelating agent (EDTA) and a step change in pH. The enzyme is a dimeric protein (Mr 83000) with two identical subunits of 41800 and contains four atoms of zinc per enzyme molecule, which are required for full activity. The enzyme follows Michaelis-Menten kinetics with Km values of 4.0 microM for folate, 8.0 microM for methotrexate and 34.0 microM for 5-methyltetrahydrofolate, the predominant form of reduced folate found in plasma.

Molecular cloning of the Pseudomonas carboxypeptidase G2 gene and its expression in Escherichia coli and Pseudomonas putida.

The gene coding for carboxypeptidase G2 was cloned from Pseudomonas sp. strain RS-16 into Escherichia coli W5445 by inserting Sau3A-generated DNA fragments into the BamHI site of pBR322. The plasmid isolated, pNM1, was restriction mapped, and the position of the gene on the 5.8-megadalton insert was pinpointed by subcloning. The expression of carboxypeptidase in E. coli was 100-fold lower than in the Pseudomonas sp. strain. When the cloned gene was subcloned into the Pseudomonas vector pKT230 and introduced into Pseudomonas putida 2440, a 30-fold increase in expression over that obtained in E. coli was observed. High expression (up to 5% soluble protein) was obtained in E. coli by subcloning a 3.1-megadalton Bg/II fragment into the BamHI site of pAT153. The increased expression was orientation dependent and is presumed to be due to transcriptional readthrough from the Tc promoter of the vector. Production of carboxypeptidase was shown to be induced (two-fold) by the presence of folic acid, and the mature protein was shown to be located in the periplasmic space of E. coli.