Rapid Spectrophotometric Differentiation Between Glutathione-Dependent and Glutathione-Independent

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1977, p Copyright C) 1977 American Society for Microbiology Vol. 34, No. 2 Printed in U.S. Rapid Spectrophotometric Differentiation Between Glutathione-Dependent and Glutathione-Independent Gentisate and Homogentisate Pathways R. L. CRAWFORD* AND T. D. FRICK University of Minnesota, Freshwater Biological Institute, Navarre, Minnesota Received for publication 8 March 1977 A total of four pathways are known for the catabolism by microorganisms of gentisate (2,5-dihydroxybenzoate) and homogentisate (2,5-dihydroxyphenylacetate). Both of these dihydric phenols can be degraded by either a glutathionedependent or a glutathione-independent reaction sequence. We found that it is not always possible to unequivocally assign glutathione dependence or independence to a particular catabolic sequence by using the well-established spectrophotometric assays at 33 nm (gentisate pathway) or 32 nm (homogentisate pathway). This paper reports a modification of the classical spectrophotometric assays that allowed an unequivocal differentiation between glutathione-dependent and glutathione-independent pathways, even when crude cell extracts contained significant quantities of cell-derived, reduced glutathione. This was accomplished by performing assays in the presence of an approximately 1-3 M solution of the sulfhydryl-binding agent N-ethylmaleimide. A number of para-dihydric phenols are Fig. 3. These more complex patterns make differentiation between possible pathways more known to be substrates for ring-fission dioxygenases (7). The two most frequently encountered examples have been gentisate (2,5-dihy- interpreted to indicate that, in some organisms, difficult. The spectral changes of Fig. 3 could be droxybenzoate) and homogentisate (2,5-dihydroxyphenylacetate). Figure 1 illustrates the pathways that exhibit both GSH-dependent one may find gentisate and homogentisate catabolic sequences described for the dissimilation of these two compounds, both of which can tively, the crude extracts that elicit these spec- and GSH-independent components. Alterna- be degraded by glutathione-dependent or glutathione-independent routes. These pathways are GSH to catalyze an apparent GSH-independent tral changes might contain enough cell-derived usually differentiated spectrophotometrically, activity against maleylpyruvate or maleylacetoacetate. since the ring-fission products (compounds II and VIII, Fig. 1) have characteristic ultraviolet-visible absorption spectra (12, 13). Figure 2 gentisate catabolism contains no rapid method The literature concerning gentisate/homo- illustrates the spectral changes that are characteristic of each of the pathways of Fig. 1. discussed in the preceeding, when spectral for differentiating between the two alternatives Crude cell extracts usually do not contain changes such as those of Fig. 3 are observed. sufficient reduced glutathione (GSH) to catalyze GSH-dependent reactions such as those identify pathway end products as accomplished One tedious method would be to rigorously shown in steps 2 and 6 of Fig. 1. To observe by Hopper et al. (1) and Crawford (3, 4). This further degradation of maleylpyruvate or maleylacetoacetate via sequences A and C (Fig. 1), stances where catabolism proceeds beyond ma- can be an involved process, particularly in in- GSH must be added to supplement reaction leate or fumarate to malate. In such a case, one mixtures. This requirement for added GSH must not only isolate the malate, but also determine its absolute stereochemical configuration usually allows an easy differentiation between GSH-dependent and GSH-independent pathways (1, 3-6, 8-1, 13). enzyme preparations by precipitating proteins (1). Alternatively, one can prepare GSH-free In our recent investigations of gentisate and with ammonium sulfate or ethanol (1, 13) or homogentisate catabolism by bacteria, we by exhaustive dialysis. These procedures, however, often irreversibly inactivate enzymes, found that spectral changes at 33 or 32 nm sometimes are not as simple as the classic patterns of Fig. 2. We have frequently encountered organisms. making their use impossible with numerous spectral changes such as those illustrated in Here we report the development of a simple, 17

2 VOL. 34, 1977 V H 2 -. OO GENTISATE/HOMOGENTISATE CATABOLISM ; C GSH * H2 ' H OH3 H HOM 3 III IV V 171 H I H s I B., 4j H2 I + 4 H3 IV VI C.H2 H2 H2 H- G1~ H 6~~- H1 OH7 H3 VIl Vill IX x D2 o H 2 H2 D. OOoH H VII Vill FIG. 1. Bacterial catabolism ofgentisate and homogentisate. I, Gentisate; II, maleylpyruvate; III, fumarylpyruvate; IV, pyruvate; V, fumarate; VI, maleate; VII, homogentisate; VIII, maleylacetoacetate; IX, fumarylacetoacetate; X, acetoacetate; 1, gentisate dioxygenase; 2, maleylpyruvate isomerase; 3, fumarylpyruvate hydrolase; 4, maleylpyruvate hydrolase; 5, homogentisate dioxygenase; 6, maleylacetoacetate isomerase; 7, fumarylacetoacetate hydrolase; 8, maleylacetoacetate hydrolase; GSH, reduced glutathione (see references 1, 4, 1, and 13 for original descriptions ofpathways C, D, B, and A, respectively). B. A C. GSH TO 1 2 FIG. 2. Spectral changes characteristic of the pathways for catabolism of gentisate and homogentisate. (A) Glutathione-dependent gentisate pathway of Fig. 1A; (B) glutathione-independent gentisate pathway of Fig. IB; (C) glutathione-dependent homogentisate pathway offig. 1C; (D) glutathione-independent homogentisate pathway of Fig. ID; (1) 1 AM GSH; (2) no GSH added. (1) and (2) refer to separate, experimental cuvettes; the increase in absorbance is due to the accumulation to maleylpyruvate (A and B) or maleylacetoacetate (C and D), and the subsequent decrease is due to the further metabolism of these compounds. A, Absorbance. x vi V.GSH O 4 GSH T i me FIG. 3. Nonclassical spectral changes sometimes observed during enzymatic degradation of gentisate and homogentisate. (A) Cell extract-catalyzed degradation of gentisate; (B) cell extract-catalyzed degradation of homogentisate. GSH was added to 1 /M. A, Absorbance. spectrophotometric procedure to differentiate gentisate or homogentisate pathways, even when crude cell extracts contain significant quantities of cell-derived GSH.

3 172 CRAWFORD AND FRICK MATERIALS AND METHODS All cultural, enzymatic, and analytical procedures were performed as previously described (2-6, 8). Solutions ofn-ethylmaleimide (NEM,.1 M) and GSH (.1 M) were freshly prepared before their use. In all spectrophotometric assays, free GSH concentrations never exceeded 1,uM (1), thereby avoiding nonenzymatic degradation of ring-fission products by GSH. Chemicals were purchased either from the Sigma Chemical Co. (St. Louis, Mo.) or the Aldrich Chemical Co. (Milwaukee, Wis.). RESULTS AND DISCUSSION When the usual spectrophotometric assays of gentisate and homogentisate degradation were performed in the presence of a 1-3 M solution of the sulfhydryl-binding agent NEM (11), it became possible to unequivocally differentiate pathways. Figure 4 illustrates the spectral changes observed at 33 rum during oxidation of gentisate by a crude extract prepared from Moraxella OA3, an organism known to degrade 2-hydroxybenzoate. by a GSH-dependent, gentisate route (6). Figure 4A illustrates spectral changes in the absence of NEM, and Fig. 4B illustrates spectral changes in the presence of 1-3 M NEM. As the figure shows, NEM completely inhibits the GSH-dependent degradation of maleylpyruvate by extracts of this organism, and this inhibition can be overcome by adding GSH in a slight excess over NEM. Similar results were obtained with an extract of Pseudomonas acidovorans, which degrades 4- hydroxyphenylacetate by a GSH-dependent, homogentisate pathway (8; Fig. 5). Figure 6 illustrates the spectral changes observed at 33 nm during degradation of gentisate by an extract prepared from Bacillus C5f, an organism known to degrade its growth substrate (3-hydroxybenzoate) via a GSH-independent, gentisate pathway (3). In this case NEM does not prevent degradation of the ringfission product, since the observed degradation of maleylpyruvate was not due to GSH present in crude cell extracts, but was independent of GSH. Similar results were obtained when extracts were prepared from Bacillus MMPA-2, an organism known to degrade 3-methoxyphenylacetate by a GSH-independent, homogentisate pathway (4; Fig. 7). The general utility of the above-described "NEM procedure" to unequivocally differentiate gentisate/homogentisate pathways is shown in Table 1. Organisms known from previous investigations to degrade gentisate and/ or homogentisate by GSH-dependent routes were examined by this technique. In all cases B..8 / ~4 ~ 4.2\ 4.2. f R FIG. 4. Effect of NEM on the degradation of gentisate by extracts of salicylate-grown Moraxella OA3 (A) in the absence ofnem and (B) in the presence of 1-3 M NEM. Reactions were performed in 1.2 ml of.1 M sodium-potassium phosphate buffer, ph 7.. At the times indicated by the arrows, the following additions were made: 1,.1 umol ofgentisate; 2, 2 pj of cell extract (.12 mg of protein) of salicylategrown OA3; 3,.1,umol ofgsh; 4,.1 Amol ofgentisate and 1.,umol ofnem; 5, 2 /1 ofcell-free extract (.12 mg of protein) of salicylate-grown OA3; 6,.1.umol of GSH; and 7, 1. uimol of GSH. Reference cuvettes received all additions except gentisate. A, Absorbance..6 N APPL. ENVIRON. MICROBIOL. B. C N LW FIG. 5. Effect of NEM on degradation of homogentisate by extracts of 4-hydroxyphenylacetategrown Pseudomonas acidovorans (A) in the absence of NEM and (B) in the presence of 1-3 M NEM. Reactions were performed in 1.5 ml of.1 M sodium-potassium phosphate buffer, ph 7.. At the times indicated by the arrows, the following additions were made: 1,.5,mol of homogentisate; 2, 5 M1 (1.5 mg ofprotein) ofcell-free extract prepared from 4-hydroxyphenylacetate-grown Pseudomonas acidovorans; 3,.1,umol of GSH; 4,.5,umol of homogentisate and 1.,umol of NEM; 5,.1,umol of GSH; 6, 1. Amol of GSH. Reference cuvettes received all additions except homogentisate. A, Absorbance. the NEM procedure gave results as illustrated in Fig. 4 for gentisate degraders or in Fig. 5 for homogentisate degraders. NEM inhibited the GSH-dependent degradation of ring-fission products and, with the exception of tyrosinegrown Moraxella OA3, this inhibition was overcome by the addition of a slight excess of GSH over NEM. Numerous organisms known to degrade gentisate and/or homogentisate by GSH-independ-

4 VOL. 34, 1977 ent pathways were also examined by using the NEM procedure (Table 1). In all cases except one (Pseudomonas strain 2,5X) this technique gave spectral patterns like those illustrated in Fig. 6 for gentisate degraders or Fig. 7 for homogentisate degraders. NEM did not prevent further degradation of ring-fission products in the absence of free GSH. Crude extracts of 3-hydroxybenzoate-grown Pseudomonas strain 2,5X (9, 1) degraded gentisate in the absence of NEM by the nonclassical pattern of Fig. 3A, indicating some GSHdependent catabolic activity. In the presence of 1-3 M NEM, portions of the same extracts degraded gentisate by the pattern of Fig. 6B, confirming the presence of a GSH-independent.6..4 M B. 6) GENTISATE/HOMOGENTISATE CATABOLISM 173 pathway in this pseudomonad as documented by Hopper et al. (1). However, on addition of a slight excess of free GSH over NEM (1.1/1.; 1 pm free GSH), the degradation rate of maleylpyruvate was increased significantly. Thus, it appears that Pseudomonas 2,5X contains both GSH-independent and GSH-dependent enzymes for the degradation of maleylpyruvate. Hopper et al. (1) reported a complete absence of a GSH-dependent maleylpyruvate isomerase in this strain. They used an ammonium sulfateprecipitated enzyme preparation. This salt treatment may have inactivated the GSH-dependent activity we observed in our crude ex- B % < Ot FIG. 6. Effect ofnem on degradation ofgentisate by extracts of3-hydroxybenzoate-grown Bacillus C5f (A) in the absence ofnem and (B) in the presence of 1-3 M NEM. Reactions were performed in 1.5 ml of sodium-potassium phosphate buffer (.1 M, ph 7.). At the times indicated by the arrows, the following additions were made: 1,.1,umol ofgentisate; 2, 5 p1 (.6 mg of protein) of cell-free extract prepared from 3-hydroxybenzoate-grown Bacillus C5f; 3,.1 g.mol of GSH; 4,.1 p.mol ofgentisate and 1.,umol of NEM. Reference cuvettes received all additions except gentisate. A, Absorbance. FIG. 7. Effect of NEM on degradation of homogentisate by extracts of 3-methoxyphenylacetategrown Bacillus MMPA-2 (A) in the absence ofnem and (B) in the presence of 1-3 M NEM. Reactions were performed in 1.5 ml of.1 M sodium-potassium phosphate buffer, ph 7.. At the times indicated by the arrows, the following additions were made: 1,.2 p1mol of homogentisate; 2, 5 (.25 mg of protein) of cell-free extract prepared from 3- methoxyphenylacetate-grown Bacillus MMPA-2; 3,.1,umol of GSH; 4,.2 p,mol of homogentisate and 1. p.mol of NEM. Reference cuvettes received all additions except homogentisate. A, Absorbance. TABLE 1. Degradation of gentisate and homogentisate by microorganisms Source of or- Reported pathway for Pathway indicated by Organism ganism or Growth substrate degradation of growth NEM procedure reference substrate Bacillus brevis Cla 3 3-Hydroxybenzoate GSH,-gentisatea GSH,-gentisate Bacillus MMPA Methoxyphenylacetate GSH,-homogentisate GSHt-homogentisate Pseudomonas acidovorans' 8 4-Hydroxyphenylacetate GSHd-homogentisatea GSHd-homogentisate Bacterium Ml New isolate 3-Hydroxybenzoate GSHd-gentisate Bacterium Ml New isolate 4-Hydroxyphenylacetate GSHd-homogentisate Pseudomonas 2,5Xc 9, 1 3-Hydroxybenzoate GSH,-gentisate GSHi,d-gentisatea Bacillus PHB-7a 5 3-Hydroxybenzoate GSH,-gentisate GSH,-gentisate Bacillus PHB-7b 5 4-Hydroxybenzoate GSH,-gentisate GSHI-gentisate Moraxella OA3 6 2-Hydroxybenzoate GSHd-gentisate GSHd-gentisate Moraxella OA3 6 L-Tyrosine GSHd-homogentisate GSHd-homogentisate Bacillus B5f 3 3-Hydroxybenzoate GSH,-gentisate GSH,-gentisate Bacillus C5f 3 3-Hydroxybenzoate GSH,-gentisate GSH,-gentisate Bacterium PHPXA New isolate L-Tyrosine GSHd-homogentisate Bacterium PHPXA New isolate 3-Hydroxybenzoate GSHd-gentisate Pseudomonas acidovoransb 8 3-Hydroxybenzoate GSHd-gentisate GSHd-gentisate a GSH,, Glutathione independent; GSHd, glutathione dependent; GSH,d both glutathione dependent and independent. b ATCC 17455, NCIB 113. c NCIB 9867.

5 174 CRAWFORD AND FRICK tracts, which were not subjected to any partial purification procedures. Two newly isolated organisms that did not give clearly defined spectral patterns (Fig. 3) were examined by the NEM procedure (Table 1). Both were easily classified as either GSH dependent or GSH independent by observing whether or not NEM caused inhibition of ringfission product degradation. NEM (1-3 M) did not significantly inhibit gentisate dioxygenase or homogentisate dioxygenase activities in crude extracts prepared from the organisms listed in Table 1. NEM also generally did not significantly inhibit enzymes that degrade maleylpyruvate, fumarylpyruvate, maleylacetoacetate, or fumarylacetoacetate. In all cases of known GSH-dependent pathways, except that of tyrosine-grown Moraxella OA3 (Table 1), addition of excess GSH (GSH/NEM = 1.1) resulted in disappearance of the ring-fission products. In all instances of the known GSH-independent pathways, ring-fission product degradation was not prevented by the presence of NEM. Further evidence to support these contentions is shown by the observation that extracts of salicylate-grown strain OA3 (Table 1) readily degrade synthetic fumarylpyruvate in the presence or absence of 1-3 M NEM. Thus, NEM does not significantly inhibit the fumarylpyruvate hydrolase of strain OA3. Another variation of gentisate catabolism has been reported, indicating that a strain of Bacillus megaterium may isomerize maleylpyruvate to fumarylpyruvate without the participation of GSH (S. R. Hagedorn, S. L. Keenan, and P. J. Chapman, Abstr. Annu. Meet. Am. Soc. Microbiol. 1977, Q86, p. 275). In such an instance, the GSH independence of the isomerization should be demonstrable by the NEM APPL. ENVIRON. MICROBIOL. procedure. The absence of significant absorption of maleylpyruvate in the near-ultraviolet region in acid solution, in contrast to the strong absorption of fumarylpyruvate under such conditions (13), should allow a demonstration of the transient accumulation of fumarylpyruvate from maleylpyruvate in the presence of NEM. The procedure described here should be of value to investigators who are examining microbial degradation of gentisate or homogentisate. In instances where degradation of maleylpyruvate or maleylacetoacetate appears to proceed by a GSH-independent mechanism, addition of approximately 1-3 M NEM to the assay mixture should not abolish this catabolic activity (Fig. 6 and 7). Where the apparent GSHindependent activity is an artifact of cell-derived GSH, addition of 1-3 M NEM will result in the accumulation of the ring-fission product (Fig. 4 and 5). ACKNOWLEDGMENTS We thank Pat Perkins for technical assistance, and the Freshwater Biological Research Foundation for building and partially equipping the Freshwater Biological Institute, which was donated 9 December 1976 by the Foundation to the University of Minnesota. In particular we thank Richard Gray, Sr., without whose efforts the Freshwater Biological Institute would not exist. This research was supported by Public Health Service grant 1RO1-ES1284-OlA1 from the National Institute of Environmental Health Sciences. LITERATURE CITED 1. Chapman, P. J., and S. Dagley Oxidation of homogentisic acid by cell-free extracts of a vibrio. J. Gen. Microbiol. 28: Crawford, R. L Novel pathway for degradation of protocatechuic acid in Bacillus species. J. Bacteriol. 121: Crawford, R. L Degradation of 3-hydroxybenzoate by bacteria of the genus Bacillus. Appl. Microbiol. 3: Crawford, R. L Degradation of homogentisate by strains of Bacillus and Moraxella. Can. J. Microbiol. 22: Crawford, R. L Pathways of 4-hydroxybenzoate degradation among species of Bacillus. J. Bacteriol. 127: Crawford, R. L., S. W. Hutton, and P. J. Chapman Purification and properties of gentisate 1,2- dioxygenase from Moraxella osloensis. J. Bacteriol. 12: Dagley, S Catabolism of aromatic compounds by microorganisms. Adv. Microb. Physiol. 6: Hareland, W., R. L. Crawford, P. J. Chapman, and S. Dagley Metabolic function and properties of 4-hydroxyphenylacetic acid 1-hydroxylase from Pseudomonas acidovorans. J. Bacteriol. 121: Hopper, D. J., and P. J. Chapman Gentisic acid and its 3- and 4-methyl-substituted homologues as intermediates in the bacterial degradation of m-cresol, 3,5-xylenol and 2,5-xylenol. Biochem. J. 122: Hopper, D. J., P. J. Chapman, and S. Dagley Enzymic formation of D-malate. Biochem. J. 11: Jocelyn, P. C Biochemistry of the SH group. Academic Press Inc., New York. 12. Knox, W. E., and S. W. Edwards The properties of maleylacetoacetate, the initial product of homogentisate oxidation in liver. J. Biol. Chem. 216: Lack, L The enzymic oxidation of gentisic acid. Biochim. Biophys. Acta 34: