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J Bacteriol. 2010 February; 192(3): 801–806.
Published online 2009 November 20. doi:  10.1128/JB.01365-09
PMCID: PMC2812461

Bypassing Isophthalate Inhibition by Modulating Glutamate Dehydrogenase (GDH): Purification and Kinetic Characterization of NADP-GDHs from Isophthalate-Degrading Pseudomonas aeruginosa Strain PP4 and Acinetobacter lwoffii Strain ISP4[down-pointing small open triangle]

Abstract

Pseudomonas aeruginosa strain PP4 and Acinetobacter lwoffii strain ISP4 metabolize isophthalate as a sole source of carbon and energy. Isophthalate is known to be a competitive inhibitor of glutamate dehydrogenase (GDH), which is involved in C and N metabolism. Strain PP4 showed carbon source-dependent modulation of NADP-GDH; GDHI was produced when cells were grown on isophthalate, while GDHII was produced when cells were grown on glucose. Strain ISP4 produced a single form of NADP-GDH, GDHP, when it was grown on either isophthalate or rich medium (2YT). All of the forms of GDH were purified to homogeneity and characterized. GDHI and GDHII were found to be homotetramers, while GDHP was found to be a homohexamer. GDHII was more sensitive to inhibition by isophthalate (2.5- and 5.5-fold more sensitive for amination and deamination reactions, respectively) than GDHI. Differences in the N-terminal sequences and electrophoretic mobilities in an activity-staining gel confirmed the presence of two forms of GDH, GDHI and GDHII, in strain PP4. In strain ISP4, irrespective of the carbon source, the GDHP produced showed similar levels of inhibition with isophthalate. However, the specific activity of GDHP from isophthalate-grown cells was 2.5- to 3-fold higher than that of GDHP from 2YT-grown cells. Identical N-terminal sequences and electrophoretic mobilities in the activity-staining gel suggested the presence of a single form of GDHP in strain ISP4. These results demonstrate the ability of organisms to modulate GDH either by producing an entirely different form or by increasing the level of the enzyme, thus enabling strains to utilize isophthalate more efficiently as a sole source of carbon and energy.

Phthalate isomers and their esters are used widely in various industries and are considered potent pollutants because of their carcinogenic, mutagenic, teratogenic, and endocrine-disrupting properties (31, 32). Due to the persistence of these compounds in the environment, microorganisms have evolved and adapted to utilize them as sole sources of carbon and energy. Compared to the organisms whose metabolic pathways for isophthalate degradation have been studied, a large number of organisms have been studied in detail to determine their metabolic pathways for phthalate and terephthalate degradation (12, 17, 18, 20, 25, 26, 29, 31, 32, 35, 36). The fewer isophthalate-degrading strains and the difficulties in isolating them could be due to the fact that isophthalate acts as a competitive inhibitor of glutamate dehydrogenase (GDH), which plays an important role at the interface of C metabolism and N metabolism (5, 11, 13, 16, 27, 28, 30, 33, 34). GDH performs oxidative deamination of glutamate to α-ketoglutarate (α-KG) and reductive amination of α-KG to glutamate, and depending on the cofactor requirement the enzyme is either NAD-, NADP-, or NAD(P)-GDH (7, 19).

Pseudomonas aeruginosa strain PP4 and Acinetobacter lwoffii strain ISP4 utilize isophthalate as a sole source of carbon and energy (31, 32). Thus, in these strains, the carbon skeleton of the glutamate family amino acids (Glu, Gln, Arg, and Pro) is derived from isophthalate. Since it is known that isophthalate is a competitive inhibitor of GDH, the question was how these bacterial strains are able to grow on isophthalate, avoid inhibition of GDH by isophthalate, and synthesize glutamate family amino acids. Carbon source-dependent NADP-GDH activities, sensitivity to inhibition by isophthalate, activity staining, and thermal stability studies suggested that (i) strain PP4 produced GDHI when it was grown on isophthalate and GDHII when it was grown on glucose and (ii) irrespective of the carbon source strain ISP4 produced only one form of GDH, GDHP (33). Since this was the first study of carbon source-dependent modulation of NADP-GDH in a bacterial system, the goal was to purify GDHI, GDHII, and GDHP and determine the biochemical and kinetic properties of these enzymes. The results obtained suggest that strain PP4 produced two GDH isoforms, GDHI and GDHII, which differed in their N-terminal sequences, sensitivity to inhibition by isophthalate, and kinetic properties, while strain ISP4 produced a single isoform, GDHP, with the same N-terminal sequence and kinetic properties when it was grown on either of the media. However, the enzyme concentration was higher (2.5- to 3-fold higher) when cells were grown on isophthalate than when cells were grown on 2YT.

MATERIALS AND METHODS

Chemicals.

HEPES, CAPS [3-(cyclohexylamino)-1-propanesulfonic acid], α-ketoglutarate, glutamate, EDTA, glycine, acrylamide, N,N′-methylene-bisacrylamide, N,N,N′,N′-tetramethylethylenediamine, DEAE-Sephacel, Phenyl-Sepharose CL-4B, Cibacron blue 3GA-agarose, and Sephacryl S300-HR were purchased from Sigma-Aldrich (United States). All other chemicals used were analytical grade and were purchased locally.

Bacterial strains and growth conditions.

P. aeruginosa strain PP4 and A. lwoffii strain ISP4, two strains capable of degrading phthalate isomers as sole carbon sources, were used in the present study (31). Strain PP4 was grown in a fermentor (working volume, 8 liters; Bioengineering, Germany) containing mineral salt medium (MSM) (2) supplemented with isophthalate (0.1%). The conditions used for the fermentor were 30°C, 400 rpm, and aeration with 7 liters min−1. Alternatively, strain PP4 was grown in MSM (150 ml) supplemented with glucose (0.25%) in 500-ml baffled Erlenmeyer flasks at 30°C on a rotary shaker (200 rpm). Strain ISP4 was grown either on MSM supplemented with isophthalate (0.1%) or on 2YT (31).

GDH assay and purification.

The enzyme activity was monitored spectrophotometrically (Perkin-Elmer, United States) by measuring the decrease in the absorbance at 340 nm as described previously (33). The reaction mixture (1 ml) contained α-KG (5 mM), ammonium chloride (200 mM), NADPH (0.1 mM), and an appropriate amount of enzyme in HEPES-NaOH buffer (100 mM, pH 8.5). The activity was expressed in micromoles of NADPH oxidized per minute. Specific activities were expressed in micromoles per minute per milligram of protein. The protein content was estimated by the method of Bradford (6) using bovine serum albumin as a standard.

The enzymes from strain PP4 (GDHI from isophthalate-grown cells and GDHII from glucose-grown cells) and strain ISP4 (GDHP from isophthalate- or 2YT-grown cells) were purified by employing heat treatment, ammonium sulfate fractionation, and ion-exchange, affinity, hydrophobic, and gel filtration column chromatography. Details of the purification procedure are described in the supplemental material.

Molecular weight determination and activity staining.

Native molecular weights of GDHs were determined using Sephacryl S300-HR gel filtration column chromatography (80 cm by 1.2 cm; bed volume, ~90 ml; void volume, 36 ml; flow rate, 5.5 ml h−1) with columns preequilibrated with filtered and degassed buffer E (20 mM HEPES-NaOH buffer [pH 8.5], 1 mM EDTA, 1 mM α-KG, 4.35% glycerol, 0.15 M sodium chloride). Subunit molecular weights were determined by discontinuous SDS-PAGE performed with resolving (12.5%) and stacking (5%) gels as described by Laemmli (21). Electrophoresis was performed using a constant current of 8 mA.

Activity staining was performed by resolving appropriate amounts of purified proteins on a native PAGE (7.5%) gel at a constant current of 4 mA at 4°C, which was followed by activity staining as described previously (33).

N-terminal sequencing.

Purified proteins were subjected to SDS-PAGE (12.5%), electroblotted onto a polyvinylidene difluoride membrane (Biodyne PVDF; 0.45 μm; Pall Corporation, United States) using CAPS-NaOH buffer (10 mM, pH 11) in 10% methanol at 100 V for 8 h, stained with Coomassie brilliant blue R-250, and subjected to automated Edman degradation (Applied Biosystems 470).

Determination of kinetic constants.

The amination reaction was monitored at the optimum pH using HEPES-NaOH buffer (100 mM, pH 8.0) for all three GDHs; the deamination reaction for GDHI and GDHII was monitored using CAPS-NaOH (100 mM, pH 10), while the deamination reaction for GDHP was monitored using glycine-NaOH (100 mM, pH 9.5). To determine the kinetic constants for the amination reaction, various concentrations of either α-KG (0.05 to 50 mM), NH4Cl (0.1 to 250 mM), or NADPH (0.0025 to 0.5 mM) and fixed concentrations of other components (5 mM α-KG, 200 mM NH4Cl, and 0.15 mM NADPH) were used. For the deamination reactions, various concentrations of either glutamate (1 to 500 mM) or NADP (0.0025 to 0.5 mM) and fixed concentration of either NADP (0.2 mM) or glutamate (100 mM for GDHI and GDHP and 200 mM for GDHII) were used. The kinetic constants Km, the inhibition constant (Ki), kcat, and kcat/Km for various substrates were determined by plotting the enzyme activities versus substrate concentrations.

Kinetics of isophthalate inhibition.

To determine the inhibition constants (Ki) for isophthalate, various fixed concentrations of isophthalate (0, 0.5, 1, 2.5, and 5 mM) were used in both the amination and deamination reactions along with various concentrations of α-KG or glutamate and fixed concentrations of other substrates, as mentioned above.

Statistical analysis and fitting of values.

All experiments were repeated at least three times, and duplicate values were obtained. Activities corresponding to substrate inhibition were plotted by using Vmax/[1 + (Km/S) + (S/Ki)], and values for mixed partial inhibition were fitted using Vmax × {[1 + β × I/(α × Ki)]/[1 + I/(α × Ki)]}/{1 + (Km/S) × (1 + I/Ki)/[1 + I/(α × Ki)]}. Standard deviations for different sets of experiments are indicated appropriately.

RESULTS

Purification of GDHI, GDHII, and GDHP.

NADP-dependent GDHI and GDHII were purified to homogeneity from P. aeruginosa strain PP4 grown on isophthalate and glucose, respectively, while GDHP was purified from A. lwoffii strain ISP4 grown on either isophthalate or 2YT. Incubation at 60°C was included as a step during purification of GDHI and GDHP (33). The enzyme yields were better in the presence of α-KG (1 mM), EDTA (1 mM), and glycerol (4.35%). GDHI and GDHII were purified 228- and 428-fold, respectively, while GDHP from cells grown on isophthalate and GDHP from cells grown on 2YT were purified 142- and 510-fold, respectively. The purification steps and profiles are summarized in Table S1 and Fig. S1 in the supplemental material. Purified enzymes were found to be stable for ~5 to 6 months at 4°C without any significant loss of activity.

GDHI, GDHII, and GDHP produced a single band at ~43 kDa on an SDS-PAGE gel (Fig. (Fig.1A;1A; see Fig. S1 in the supplemental material), as well as on a native PAGE gel (Fig. 1B and C). Using Sephacryl S300-HR gel filtration chromatography, the native molecular masses of GDHI, GDHII, and GDHP were determined to be ~170, ~170, and ~250 kDa, respectively (Fig. (Fig.1D).1D). These results suggested that GDHI and GDHII were homotetramers, while GDHP was a homohexamer.

FIG. 1.
Molecular properties of GDHI, GDHII, and GDHP. (A) SDS-PAGE profile of purified GDHs. Lane 1, GDHP from 2YT-grown cells; lane 2, GDHP from isophthalate-grown cells; lane 3, GDHII; lane 4, GDHI. Lane M contained molecular mass standards, including phosphorylase ...

Activity staining and N-terminal sequencing.

GDHI, GDHII, and GDHP produced a single activity-staining band on a PAGE gel. Mixing of purified GDHI and GDHII resulted in two distinct activity-staining bands (Fig. (Fig.1B),1B), while a single band was observed when GDHP preparations purified from isophthalate- and 2YT-grown cells were mixed and electrophoresed (Fig. (Fig.1C).1C). The N-terminal sequence of GDHI was different from that of GDHII, while GDHP purified from isophthalate-grown strain ISP4 and GDHP purified from 2YT-grown strain ISP4 had the same sequence (Table (Table1).1). The N-terminal sequence of GDHI did not show any similarity to known NADP-GDH sequences, while GDHII showed sequence similarity to the N termini of NADP-GDHs from P. aeruginosa strains PA7 and PAO1 (Table (Table1).1). The N terminus of GDHP showed similarity to the sequences of Acinetobacter bowmanii strain SDF and AYE GDHs (Table (Table11).

TABLE 1.
N-terminal sequences of GDHI, GDHII, and GDHP and comparison with previously described GDHs

Kinetic characterization.

All three purified enzymes showed amination and deamination activities in the presence of NADPH and NADP, respectively, but not in the presence of NADH and NAD (data not shown). Both GDHI and GDHII showed pH optima of 8.0 and 10.0 for amination and deamination, respectively, while GDHP from either isophthalate- or 2YT-grown cells showed pH optima of 8.0 and 9.5 for amination and deamination, respectively.

The kinetic constants (Km, Ki, kcat, kcat/Km, and Hill coefficient [nH]) for GDHI, GDHII, and GDHP with α-KG, glutamate, NH4Cl, NADPH, and NADP were determined and are summarized in Table Table2.2. GDHI, GDHII, and GDHP showed Michaelian kinetics only with NADP (see Fig. S2 to S5 in the supplemental material) and showed substrate inhibition with α-KG at concentrations greater than 5 mM (Fig. (Fig.2A2A and Table Table2)2) and with 200 μM NADPH (Fig. (Fig.2B2B and Table Table2).2). However, the three enzymes differed with respect to the concentration required for glutamate inhibition. GDHI and GDHP showed inhibition with glutamate at concentrations greater than 100 mM, while GDHII showed inhibition with 200 mM glutamate (Table (Table2;2; see Fig. S2 to S5 in the supplemental material). All enzymes showed low Km values for α-KG compared to the Km values for glutamate (Table (Table2).2). GDHP from strain ISP4 grown on isophthalate and GDHP from strain ISP4 grown on 2YT had similar kinetic constants (Table (Table2;2; see Fig. S4 and 5 in the supplemental material).

FIG. 2.
Kinetic properties of GDH. (A) Plot of different concentrations of α-KG versus the activity profile of GDHI. Similar inhibition profiles were observed for GDHII and GDHP. (B) Plot of different concentrations of ΝΑDPH versus the ...
TABLE 2.
Kinetic constants of NADP-GDHs from strains PP4 and ISP4 grown on either isophthalate or glucose (2YT)a

When NH4Cl was one of the substrates, GDHI, GDHII, and GDHP showed two different Km values at lower and higher concentrations and apparent negative cooperativity with the Hill coefficient (nH) values below unity (Fig. (Fig.2C2C and Table Table2;2; see Fig. S2 to S5 in the supplemental material). The downward curve in the reciprocal plot at the higher NH4+ concentration revealed an enzyme with a decreased affinity for NH4Cl, a feature of negative cooperativity (Fig. (Fig.2C2C and Table Table2;2; see Fig. S2 to S5). Extrapolation of kinetic constants at the lower and higher concentrations of ammonia revealed ~7- to 14-fold increases in the Km (Table (Table2)2) and ~2- to 3-fold increases in the Vmax (data not shown) for all three GDHs.

Inhibition with isophthalate.

GDHI, GDHII, and GDHP showed a mixed partial type of inhibition with isophthalate in both the amination and deamination reactions (Fig. (Fig.3;3; see Fig. S6 in the supplemental material). The inhibition constants obtained with isophthalate are summarized in Table Table3.3. The Ki values for GDHI were 1.75 and 5.5 times higher than those for GDHII in the amination and deamination reactions, respectively, indicating that GDHII has more affinity for isophthalate than GDHI has; also, compared to the amination reaction, the deamination reaction was more sensitive to inhibition (Table (Table3).3). The α factor for GDHII was ~1.5 times higher than that for GDHI, suggesting that the rate of conversion of the GDHII-α-KG/glutamate-isophthalate (ESI) complex was lower for GDHII. The observed β values were insignificant for most of the enzymes (Table (Table3).3). The inhibition constants for GDHP from strain ISP4 grown on isophthalate and GDHP from strain ISP4 grown on 2YT were similar, and the deamination reaction (Ki and α factor) was more sensitive to isophthalate than the amination reaction (Table (Table3;3; see Fig. S6 in the supplemental material).

FIG. 3.
Double-reciprocal plot of velocity (v) versus α-KG concentration for GDHII in the presence of different concentrations of isophthalate and fixed concentrations of NADPH (150 μM) and NH4Cl (200 mM) in HEPES-NaOH buffer (100 mM, pH 8.0). ...
TABLE 3.
Inhibition constants for NADP-GDHs from strains PP4 and ISP4 with isophthalatea

DISCUSSION

Many organisms have been reported to produce different forms of GDH under various conditions like salt, cold, trophic, or environmental stress (4, 8, 24). Interestingly, P. aeruginosa strain PP4 and A. lwoffii strain ISP4 showed carbon source-dependent modulation of NADP-GDH, leading to expression of different forms or levels of the same enzyme. To the best of our knowledge, this is the first report for a bacterial system.

GDHI from isophthalate-grown cells and GDHII from glucose-grown cells of strain PP4 and GDHP from isophthalate- and 2YT-grown cells of strain ISP4 were purified to homogeneity. Compared to GDHII, GDHI and GDHP were found to be heat stable; therefore, heat treatment of cell extract at 60°C was employed as one of the purification steps. Thermostable GDHs obtained from mesophilic organisms have been reported for Chlamydomonas reinhardtii (24), Peptostreptococcus asaccharolyticus (9), Pseudomonas sp. strain AM1 (3), and Clostridium symbiosum (38); however, the significance of this is not clear. Based on SDS-PAGE and gel filtration analyses, GDHI and GDHII were homotetramers, while GDHP was a homohexamer with a subunit molecular mass of 43 kDa, which is similar to the subunit molecular masses of most of the reported NADP-GDHs from different organisms (19). However, the molecular properties of GDHI were distinctly different from those of GDHII, as the two enzymes had distinct N-terminal sequences and produced distinct activity-staining bands. The N-terminal sequence of GDHII was similar to the sequences of P. aeruginosa strains PA7 and PAO1 reported in the genome data bank. However, the molecular properties of the enzymes and the conditions under which they are expressed are not clear. The N-terminal sequence of GDHI did not match any of the known sequences of GDHs in the data bank, suggesting that this enzyme is unique. The molecular properties of the GDHP preparations from isophthalate- and 2YT-grown cells of strain ISP4 were identical. However, as shown by the specific activity and purification data, the level of expression was about four times higher for the GDHP from cells grown on isophthalate than for the GDHP from 2YT-grown cells.

GDHI, GDHII, and GDHP showed higher affinity for α-KG than for glutamate, and the NADP dependence indicated that the enzymes were anabolic. Higher concentrations of glutamate, α-KG, and NADPH inhibited the enzymes. Similar inhibition kinetics were observed for other GDHs and were proposed to be a regulatory feature (15, 19, 23). Inhibition with NADPH was an interesting feature and was reported previously for a few GDHs (14, 22, 23). Based on studies with bovine GDH, this property could be due to the presence of an additional coenzyme binding regulatory site, or the substrates and NADPH might not exhibit ordered binding to the enzyme at very high concentrations of NADPH (1, 19, 22). GDHs from strains PP4 and ISP4 showed biphasic saturation kinetics with NH4Cl, similar to other GDHs (10, 14, 15, 23, 37). A plausible reason for this is the presence of both low- and high-affinity sites on the enzyme that operate independently depending on the cellular concentration of ammonia or the presence of various conformational states of a multisite enzyme that slowly equilibrate in the presence of various ligands and physical agents (15, 23). The occurrence of multiple kinetic populations of the enzyme might be due to inherent asymmetry in the tetrameric or hexameric structure. However, these explanations require further experimental validation.

Interestingly, GDHs from strains PP4 and ISP4 showed a mixed partial type of inhibition with isophthalate, suggesting that isophthalate binds not only to the enzyme form but also to the enzyme-substrate form. The inhibition constants (Ki) with isophthalate suggested that deamination reactions were far more sensitive to inhibition than amination reactions, as observed with several GDHs (5, 13, 16, 27, 28, 30, 34). The Ki values suggested that compared to GDHI, GDHII has a higher affinity for isophthalate, thus making it more susceptible to inhibition. The α factor of GDHII was ~1.5 times higher than that of GDHI, indicating that the ESI complex (GDHII-α-KG/glutamate-isophthalate) was more stable and the rate of conversion of this complex was lower with GDHII than with GDHI. Even though the inhibition observed was mixed partial, the β values were insignificant. Thus, the presence of GDHI in strain PP4, which is less susceptible to inhibition by isophthalate, provides an advantage to the organism for better survival and growth on isophthalate as a carbon source. As observed for GDHII, the affinity of GDHP for isophthalate was higher for the deamination reaction than for the amination reaction. The rate of conversion of the ESI complex was ~10 to 14 times lower when α-KG was used as a substrate than when glutamate was used as a substrate. GDHII and GDHP were more sensitive to inhibition by isophthalate than GDHI.

These results demonstrate that strains PP4 and ISP4 have different strategies to overcome the inhibitory effect of isophthalate when it is used as a sole source of carbon and energy. When grown on isophthalate, strain PP4 produces the unique enzyme GDHI, which is less sensitive to inhibition by isophthalate than GDHII. On the other hand, strain ISP4 produces only one form of GDHP, whose inhibition by isophthalate is counteracted by increased levels when the organism is grown on isophthalate. The strategies used are different and unique to these strains. Thus, understanding the different strategies that these strains use to overcome the inhibitory metabolic effect of a sole carbon source, in addition to the basic metabolism and enzymology of isophthalate degradation, should help workers to use these organisms for effective bioremediation.

Supplementary Material

[Supplemental material]

Acknowledgments

P.S.P. thanks the Department of Biotechnology, Government of India, for providing a research grant. V.K.C. acknowledges a senior research fellowship from CSIR, Government of India.

P.S.P. and V.K.C. thank N. S. Punekar, Department of Biosciences and Bioengineering, IIT-Mumbai, for constructive suggestions and discussions.

Footnotes

[down-pointing small open triangle]Published ahead of print on 20 November 2009.

Supplemental material for this article may be found at http://jb.asm.org/.

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