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Purification and characterization of o-hydroxyphenylacetate 5-hydroxylase, m-hydroxyphenylacetate 6-hydroxylase and p-hydroxyphenylacetate 1-hydroxylase from Rhodococcus erythropolis
JOURNAL OFFKRMENTATION ANDBIOENOINEERING Vol. 81, No. 2, 133-137. 1996
Purification and Characterization of o-Hydroxyphenylacetate SHydroxylase, m-Hydroxyphenylacetate 6-Hydroxylase and p-Hydroxyphenylacetate 1-Hydroxylase from Rhodococcus erythropolis AK10 SUEMORI,*
KENJI NAKAJIMA,
RYUICHIRO
National Institute of Bioscience and Human-Technology
KURANE,
AND YOSHIHIRO
NAKAMURA
(NIBH), I-i Higashi, Tsukuba, Ibaraki 305, Japan
Received 27 July 1995/Accepted 13 November 1995 The gram-positive Rhodococcus erythropolis strain Sl was found to utilize o-, m-, and p-hydroxyphenylacetic acids as sole carbon sources. Each isomer of monohydroxyphenylacetate was degraded via the homogentisate pathway, which contained a reduced glutathione independent-isomerase. Three monohydroxyphenylacetate monooxygenases, o-hydroxyphenylacetate 5-hydroxylase, m-hydroxyphenylacetate B-hydroxylase, and p-hydroxyphenylacetate 1-hydroxylase, were purified to homogeneity from strain Sl. Each enzyme was a 45kDa monomeric NADH-dependent monooxygenase containing FAD, and all three appeared to belong to the p-hydroxybenzoate hydroxylase-class as regards the flavin-containing aromatic compound monooxygenase family. However, the three enzymes differed greatly in terms of substrate specificity. [Key words: Rhodococcus erythropolis, o-hydroxyphenylacetate hydroxylase, p-hydroxyphenylacetate I-hydroxylase]
6-
Sigma (USA). Sodium salicylate, sodium m-hydroxybenzoate, sodium p-hydroxybenzoate, gentisic acid, and protocatechuic acid were purchased from Wako Pure Chemical Company (Osaka). Bacterium and media R. erythropolis strain Sl was isolated from soil with an enrichment method using a phthalate ester as sole carbon source (13). YMG medium consisted of 4 g of yeast extract (Difco), 10 g of malt extract (Difco), and 4 g of glucose in 1 1 of distilled water, pH 7.3. The minimal medium composition was the same as described previously (14). Strain Sl was preincubated in YMG medium with shaking at 30°C for 1 d. One ml of YMG-preculture was inoculated into 1OOml of minimal medium at O.l-O.3% monohydroxyphenylacetate and incubated with shaking at 30°C for 16 h. Cells in the logarithmic growth period were harvested by centrifugation and then washed twice in distilled water. They were then either used immediately in an oxygen uptake experiment or stored at -80°C until use for enzyme purification. Oxygen uptake The assay system for oxygen uptake by intact cells grown on aromatic compounds contained a suitable amount of washed cells and 2 mM substrate in a final volume of 2 ml of 10 mM sodium phosphate buffer, pH 7.1 (10). The oxygen consumption rate (OCR) was monitored using a biological oxygen monitor (YSI model 5300). As the control, the assay system, omitting substrate, was measured and OCR compared with the aromatic compound was defined by reducing the OCR of the substrate-omitted system from that of the complete assay system. Homogentisate pathway For characterization of the homogentisate pathway, a crude enzyme solution was prepared as follows. Frozen cells grown on 0.3% MHPA were suspended in a 10mM sodium phosphate buffer, pH 7.1, containing 10% (v/v) glycerol and disrupted by sonication for 18 min. The mixture was centrifuged (2O,OOOxg, 60min) and ammonium sulfate was added to the supernatant to 30% saturation. The crude
Many microorganisms have been shown to degrade aromatic compounds in the environment, but the mechanism of degradation of aromatic compounds by gram-positive bacteria and actinomycetes is not yet fully understood. In addition, the metabolism of phenylacetates and monohydroxyphenylacetates by microorganisms has been studied extensively (l-6), while little attention has been given to the functions of monohydroxyphenylacetate hydroxylases belonging to the flavin-containing aromatic compound monooxygenase family (4-6). The gram-positive Rhodococcus erythropolis strain Sl is capable of assimilating not only various aromatic carboxylic acids such as monohydroxybenzoates (7), dihydroxybenzoates (8, 9), and phthalate (10) but also aromatic amino acids (11). L-Tyrosine may also be degraded via formation of p-hydroxyphenylacetate and homogentisate by strain S 1 (12). Our attention has thus been directed to the degradation of monohydroxyphenylacetates using strain Sl. The degradation of o-, m-, and p-hydroxyphenylacetates using strain St and characterization of the three monohydroxyphenylacetatebreakdown pathways involving the homogentisate pathway are discussed in this report. Here, we also report on the purification and some of the properties of three monohydroxyphenylacetate monooxygenases, o-hydroxyphenylacetate 5-hydroxylase (OHPASH), m-hydroxyphenylacetate 6-hydroxylase (MHPA6H), and p-hydroxyphenylacetate 1-hydroxylase (PHPAlH), from strain Sl. MATERIALS
S-hydroxylase, m-hydroxyphenylacetate
AND METHODS
Materials o-Hydroxyphenylacetic acid (OHPA), mhydroxyphenylacetic acid (MHPA), p-hydroxyphenylacetic acid (PHPA), homogentisic acid (i.e. 2,5dihydroxyphenylacetic acid), and homoprotocatechuic acid (i.e. 3,4-dihydroxyphenylacetic acid) were obtained from * Corresponding author. 133
134
J. FERMENT. BIOENG.,
SUEMORI ET AL.
extract was applied to a Butyl-Toyopearl 650M column equilibrated with the buffer 30% saturated with ammonium sulfate. After thoroughly washing the column with the same buffer to remove various elements such as NAD(P)H, the enzymes were eluted with the buffer without any ammonium sulfate. The major protein fraction was used for the enzyme assay and characterization of the homogentisate pathway. An enzyme reaction system containing 0.2mg of protein and 0.05 mM homogentisate in a final volume of 2 ml of 50mM HEPES/NaOH buffer, pH 8.0, at 24°C was used (15). The reaction was monitored spectrophotometrically based on the time course of absorbance at between 330 and 345 nm. For examination of the effects of reduced glutathione (GSH) or N-ethylmaleimide (NEM), GSH or NEM added to the reaction mixture to a final concentration of 25 mM (16). Enzyme assay The assay system for the monohydroxyphenylacetate monooxygenase activity contained a suitable amount of enzyme, 0.1 mM NADH, and 0.5 mM OHPA, MHPA, or PHPA in a final volume of 2 ml of 10 mM HEPES/NaOH buffer, pH 8.0. The enzyme activity was measured spectrophotometrically by measuring the decrease in the absorbance at 340 nm with oxidation of NADH. One unit of activity was defined as the amount of the enzyme that catalyzed the oxidation of NADH at a rate of 1 ,nmol per min at 30°C. Assay systems for the activity of homogentisate 1,2-dioxygenase (17), gentisate 1,Zdioxygenase (18), or protocatechuate 3,4-dioxygenase (19) each contained a suitable amount of enzyme, 0.5 mM homogentisate, gentisate, or protocatechuate, and 0.05 mM Fez+ in a final volume of 2 ml of 1OmM TAPS/NaOH buffer, pH 8.5. Enzyme activity was measured spectrophotometrically based on the increase in the absorbance at 330, 334, or 340nm, determined by the change in the amounts of maleylacetoacetate, maleylpyruvate, and P-carboxy-ciscis-muconate. One unit of activity was defined as the amount of enzyme catalyzing enzyme production in each case at a rate of 1 /*mol per min at 30°C. Purification and characterization of enzymes OHPASH, MHPA6H, and PHPAlH were purified from cell-free extracts of OHPA-, MHPA-, and PHPA-grown cells of strain Sl, respectively, as follows. All steps were carried out at below 5°C. The buffer used in all steps consisted of 10% (v/v) glycerol in a 10 mM sodium phosphate buffer, pH 7.0. Protein content was calculated from the absorbance of enzyme solutions at 260 and 280 nm. Step I. Crude extract Frozen cells (approximately 10 g) were suspended in a buffer and disrupted by sonication for 15 min. The turbid mixture was centrifuged (2O,OOOxg, 60min), and the resultant supernatant was used as the crude extract. Ammonium sulfate was added TABLE 1.
to the crude extract to 30% saturation. Step 2. Chromatography on Butyl- Toyopearl6SOM The enzyme solution was applied to a Butyl-Toyopearl 650M column equilibrated with a buffer 30% saturated with ammonium sulfate. After the column was washed with the same buffer, the enzyme was eluted with buffer with a linear 30-10% ammonium sulfate saturation gradient at 1.5 ml per min. The active fractions were collected and ammonium sulfate was added to the enzyme solution to obtain a saturation of 70%. The solution was left for 1 d. After centrifugation at 20,000~ g for lOmin, the precipitate was dissolved minimum volume of buffer. Step 3. Gel filtration on Toyopearl HW-55F The enzyme solution was applied to a Toyopearl HW-55F column equilibrated with buffer, and eluted with the same buffer at 0.38 ml per min. The active fractions were combined. Step 4. Chromatography on DEAE-Toyopearl65OS The enzyme solution was applied to a DEAE-Toyopearl 650s column equilibrated with the buffer. After the column was washed with the same buffer, the enzyme was eluted with the buffer with a linear O-O.3 M NaCl gradient at 0.38 ml per min. The active fractions were pooled and used for analysis of the properties of the purified enzyme. The purity of the enzyme at each purification step was assayed by native PAGE. This was carried out using a linear 5-20% polyacrylamide gel gradient in 0.025 M Tris and 0.192 M glycine buffers. The gel was stained with Coomassie brilliant blue G-250. The molecular weight of each enzyme was estimated by gel filtration using a Toyopearl HWSSF column. The column was equilibrated with a 10mM sodium phosphate buffer, pH 7.0, with 0.1 M NaCl. As reference proteins, ,%galactosidase (M, 540,000), catalase (Mr 244,000), bovine serum albumin dimer (Mr 130,000), peroxidase (Mr 44,000), and cytochrome c (M, 13,000) were used. The molecular weight of each enzyme was also estimated using SDS-PAGE with a linear S-20% polyacrylamide gel gradient in a 0.025 M Tris and 0.192 M glycine buffer with 0.1% SDS. Myosin (Mr 212,000), az-macrogloblin (Mr 170,000), $-galactosidase (Mr 116,000), transferrin (IV& 76,000), and glutamic dehydrogenase (Mr 53,000) were used as the standard marker proteins. The fluorescence of the enzyme was measured using the enzyme solution dialyzed completely against the 10 mM sodium phosphate buffer, pH 7.0. RESULTS Growth on three monohydroxyphenylacetates Strain Sl grew on OHPA, MHPA, or PHPA as sole carbon source (data not shown). Doubling times on the sub-
Oxygen consumption rate with monohydroxyphenylacetates and dihydroxyphenylacetates by intact cells of R. erythropolis Sl grown on monohydroxyphenylacetates, homogentisate, or YMG medium Oxygen consumption rate (nmol/rnin/ml/0.D.660)
Substrate
OHPA-grown
OHPA MHPA PHPA Homogentisate Homoprotocatechuate
81 9 10 96 4
MHPA-grown 8 71 12 138 3
PHPA-grown 3 11 74 120 6
Homogentisate-grown 9 13 16 173 7
YMG medium-grown 4 6 7 9
1
OHPA, MHPA, and PHPA-grown 31 36 40 106 2
VOL. 81, 1996
PURIFICATION
TABLE 2. Assay of enzyme activity in crude extracts prepared from cells of R. eryfhropofis Sl grown on OHPA, MHPA, or PHPA Enzyme activity assayed
Enzyme activity (Wmg-protein) OHPA-grown MHPA-grown PHPA-grown
OHPASH 2.8 MHPABH 0.3 PHPAlH 0.2 SALSH
0.4 3.1 0.2
0.1 0.3 4.0
strates were essentially the same, though with OHPA or PHPA, a lag time for 12-24 h was observed. Also, strain Sl grew on homogentisate and homoprotocatechuate as sole carbon sources. Induction of enzymes OCR compared against the monohydroxyphenylacetates and dihydroxyphenylacetates by intact cells grown on 0.2% (w/v) monohydroxyphenylacetate, 0.2% (w/v) dihydroxyphenylacetate, or YMG medium was measured (Table 1). Cells that were grown on OHPA demonstrated full oxygen uptake with OHPA and homogentisate. When grown with MHPA, the full oxygen uptake of MHPA and homogentisate could be observed whereas cells grown on PHPA showed full oxygen uptake with PHPA and homogentisate. Cells grown on OHPA, MHPA, and PHPA failed to have any oxygen uptake with homoprotocatechuate. Those grown on homogentisate showed almost only homogentisate after being oxidized. Cells grown in YMG medium failed to oxidize OHPA, MHPA, PHPA, or homogentisate. All three isomers thus appear to undergo hydroxylation to homogentisate due to strain Sl and the oxidation of each monohydroxyphenylacetate to be induci-
OF AROMATIC COMPOUND MONOOXYGENASES
135
ble by use of an appropriate monohydroxyphenylacetate as the growth substrate. On the other hand, after the cells were incubated in minimal medium containing all three isomers, the oxygen uptake with all three isomers by intact cells was observed (Table 1). The crude extract from cells grown on OHPA, MHPA, or PHPA was prepared and examined with respect to various enzyme activities (Table 2). Crude extracts from cells grown on OHPA showed NADH-oxidizing activity only in the presence of OHPA. Crude extracts from MHPA- or PHPA-grown cells exhibited enzyme activity only in the presence of MHPA or PHPA, respectively. It thus appears that each of the three enzymes may be involved in hydroxylation of the three monohydroxyphenylacetates, respectively. The enzymes were designated as o-hydroxyphenylacetate 5-hydroxylase, OHPASH, hydroxylating OHPA to homogentisate; m-hydroxyphenylacetate 6-hydroxylase, MHPA6H, hydroxylating MHPA to homogentisate; and p-hydroxyphenylacetate 1-hydroxylase, PHPAlH, catalyzing the hydroxylation and decarboxylation of PHPA to homogentisate (Fig. 1). All hydroxylation reactions shown by crude extracts a strict preference for NADH. The crude extracts from cells grown on the three monohydroxyphenylacetate isomers expressed full homogentisate 1,2dioxygenase activity. There was hardly any gentisate 1,2dioxygenase or protocatechuate 3,4-dioxygenase activity. Thus, we concluded that in the degradation of monohydroxyphenylacetates, only the homogentisate pathway was induced and used. GSH-independent bomogentisate pathway In the homogentisate pathway characterization, a shift in the absorbance maximum from 330 to 345 nm could be observed, indicating conversion of maleylacetoacetate to fumarylacetoacetate as a result of isomerase. A decrease in absorbance at 330 nm was not promoted by GSH, nor was it inhibited by NEM in the presence of GSH (16).
COOH
0
OH
&
(1)
\
cFHydroxyphenylacetate
mHydroxyphenylacetate
Homogentisate
Maleylacetoacetate
Fumarylacetoacetate
COOH
4 0
/
(3)
pHydroxyphenylacetate FIG. 1. Degradative pathways of OHPA, MHPA and PHPA by R. erythropolis Sl. (1). o-Hydroxyphenylacetate 5-hydroxylase; (2), mhydroxyphenylacetate 6-hydroxylase; (3), p-hydroxyphenylacetate I-hydroxylase; (4), homogentisate 1,2-dioxygenase; (5), GSHindependent maleylacetoacetate isomerase.
136
J. FERMENT.BIOENG.,
SUEMORI ET AL. TABLE 3.
Summary of purification procedures of OHPASH, MHPA6H, and PHPAlH OHPASH
Steps
Specific activity (U/m& Crude extract 12 Butyl-Toyopearl 192 Gel filtration 576 DEAE-Toyopearl 2100
Yield (%) 100 78 49 21
MHPA6H Purification (fold) 1 16 48 175
Specific activity (v/m& 16 176 1104 2544
Similar findings were observed for the pathway of Bacillus sp. (20). The relationship of the time course of 330 and 345 nm was virtually the same as that for the pathways involving the GSH-independent maleylacetoacetate isomerase (21). Accordingly, the following ring-cleavage of homogentisate by homogentisate 1,Zdioxygenase in strain Sl, maleylacetoacetate may be converted to fumarylacetoacetate due to GSH-independent maleylacetoacetate isomerase activity (Fig. 1). Homogentisate was also found to be degraded via the GSH-independent homogentisate pathway in strain Sl, which is in good agreement with findings on the GSH-independent maleylacetoacetate isomerases in some gram-positive microorganisms (19-21). Strain Sl was previously shown to possess the GSH-independent gentisate pathway (15). All the results of the present study are consistent with the fact that gram-positive bacteria possess a GSHindependent (homo)gentisate pathway (21). Puritication and characterization of three enzymes The purification procedures for the three enzymes, OHPASH, MHPA6H, and PHPAlH, are summarized in Table 3. Each of the three purified enzymes gave a single protein band on SDS-PAGE (data not shown). OHPASH, MHPA6H, and PHPAlH were purified approximately 175, 159, and 194-fold with 21, 14, and 19% activity yield, respectively. The molecular weights of OHPASH, MHPA6H, and PHPAlH were estimated to be 49, 50, and 49 kDa by gel filtration but were all estimated to be 45 kDa by SDS-PAGE. Each native enzyme thus appears to be a monomer. As fluorescence examination, after emission at 520 nm, the excitation spectrum showed their maxima at 375 nm and 454 nm. When excited at 450 nm, an emission maximum was observed at 520nm. Each spectrum was in good agreement with that of FAD. The three enzymes were thus concluded to be flavin-containing aromatic compound monooxygenases. The activity of each enzyme was examined using each TABLE 4.
Substrate specificities of OHPASH, MHPA6H, and PHPAlH from R. erythropolis Sl
Substrate o-Hydroxyphenylacetate m-Hydroxyphenylacetate p-Hydroxyphenylacetate Sahcylate m-Hydroxybenzoate p-Hydroxybenzoate o-Hydroxyphenoxyacetate m-Hydroxyphenoxyacetate p-Hydroxyphenoxyacetate p-Hydroxypropionate n-(BenzylalcohoBacetate NT, Not tested.
Relative activity (%) OHPASH MHPA6H PHPAlH 100 <5 <5 6 NT NT 26 NT NT NT NT
from R. erythropolis Sl
<5
<5
100 <5 NT <5 NT NT 19 NT NT NT
<5 100 NT NT <5 NT NT 32 28 <5
Yield (%) 100 86 30 14
PHPAlH Purification (fold) 1 11 69 159
Specific activity (U/mg) 14 280 1316 2716
Yield (%) 100 73 24 19
Purification (fold) 1 20 94 194
of several aromatic compounds as a substrate (Table 4). OHPASH oxidized mainly OHPA and to some extent ohydroxyphenoxyacetate. MHPA6H showed the strongest activity toward MHPA and some toward m-hydroxyphenoxyacetate. PHPAlH reacted with PHPA and to a slight degree with p-hydroxyphenoxyacetate and phydroxyphenylpropionate. Thus, the substrate specificities of the enzymes varied greatly. On the other hand, all enzymes were specific for NADH; i.e., they showed no activity toward NADPH. DISCUSSION These results support the view that all three isomers of monohydroxyphenylacetate are converted by hydroxylation to homogentisate by strain Sl, which produces these three enzymes that clearly differ from monohydroxybenzoate hydroxylases such as salicylate 5-hydroxylase (SALSH), m-hydroxybenzoate 6-hydroxylase (MHB6H), and p-hydroxybenzoate 3-hydroxylase (PHB3H) (7). All three monohydroxyphenylacetate hydroxylases from strain Sl were shown to be 45-kDa monomers, which is in good agreement with the subunit size of PHPAlH from Pseudomonas acidovorans (3) and 4-hydroxyphenylacetate 3-hydroxylase (PHPA3H) from Pseudommonas putida (4), as well as SALSH, MHB6H, and PHB3H from strain Sl (7). These data strongly suggest that the three monohydroxyphenylacetate hydroxylases from strain Sl belong to the p-hydroxybenzoate hydroxylase class of enzymes which have a 45-kDa subunit (6). Furthermore, all three monohydroxyphenylacetate hydroxylases from strain Sl were shown to have strong preference for NADH as did the three monohydroxybenzoate hydroxylases from the same strain (7). The co-substrate specificities of purified monohydroxyphenylacetate hydroxylases differ somewhat. For instance, MHPA6H from Flavobacterium sp. JS-7 (5) and PHPAlH from P. acidovorans (3) use NADH and NADPH with essentially the same efficiency. However, PHPAlH from Klebsiella pneumoniae (2) and PHPA3H from P. putida (4) display strong NADH preference. It is quite interesting that one bacterial strain produces various inducible flavin-containing aromatic compound monooxygenases such as OHPASH, MHPABH, PHPAlH, SALSH, MHB6H, and PHB3H having the same subunit sizes but narrow substrate specificity. Most flavin-containing aromatic compound monooxygenases catalyze the incorporation a new hydroxyl group in ortho position onto an existing hydroxyl group (21); however, all three monohydroxyphenylacetate hydroxylases from strain Sl catalyze para hydroxylation, as do SALSH and MHB6H. In consideration of the mode of hydroxylation of monohydroxylated aromatic rings, OHPASH, MHPA6H, and PHPAlH appear functionally more similar to SALSH and MHB6H than PHB3H in strain S 1.
PURIFICATION
VOL. 81, 1996
In consideration of the patterns of the inducement of oxidative activity for monohydroxyphenylacetates, for example, MHPA-grown cells of Flawbacterium sp. JS-7 could oxidize both MHPA and PHPA, while PHPAgrown cells showed a very low level of oxidative activity with MHPA (1). Washed cells of K. pneumoniae M5al grown on MHPA or PHPA oxidized MHPA and PHPA (2). These findings are somewhat at a variance with those on the effects of the growth substrate on the induction of MHPA6H or PHPAlH in strain Sl. The manner in which monohydroxyphenylacetate degradative pathways in microorganisms as well as monohydroxybenzoate degradative pathways are regulated and may not be the same in all cases (15). It is considered that m-hydroxybenzoate and p-hydroxybenzoate are catabolite repressors and may affect the level of the oxidative activity of salicylate in strain Sl, however, repression by other monohydroxyphenylacetate isomers was not observed in this study during the growth of strain Sl on monohydroxyphenylacetates. The three monohydroxyphenylacetate hydroxylases and three monohydroxybenzoate hydroxylases from strain Sl may be derived from a common ancestral protein/gene, and thus, their physicochemical and genetic properties should be carefully examined, REFERENCES I
2,
3.
4.
5.
6.
van den Tweel, W. J. J., bits, J. P., and de Boot, J. A.M.: Catabolism of DL-a-phenylhydracrylic, phenylacetic and 3- and 4-hydroxyphenylacetic acid via homogentisic acid in a Fluvobacterium sp.-Arch. Microbial., 149, 207-213 (1988). Martin. M.. Gibello. A., FemandCz. J.. Ferrer. E.. and GarridoPertieria, A.: Catadolis’m of 3- and 4-hydrox$ph;nylacetic acid by Klebsiella pneumoniae. J. Gen. Microbial., 132, 621-628 (1991). Hareland, W. A., Crawford, R. L., Chapman, P. J., and DagIey, S.: Metabolic function and properties of 4-hydroxyphenylacetic acid 1-hydroxylase from Pseudomonas acidovorans. J. Bacterial.. 121. 272-285 (1975). Raju, S. G., Barn&, A. V., and iaidyanathan, C. S.: Purification and properties of 4-hydroxyphenylacetic acid 3-hydroxylase from Pseudomonas putidu. Biochem. Biophys. Res. Commun., 154, 537-543 (1988). van Berkel, W. J. H. and van den Tweel, W. J. J.: Purification and characterization of 3-hydroxyphenylacetate 6-hydroxylase: a novel FAD-dependent monooxygenase from a Flavobacteriurn species. Eur. J. Biochem., 201, 585-592 (1991). Harayama, S., Kok, M., and Neidle, E. L.: Functional and evolutionary relationships among diverse oxygenases. Annu.
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Rev. Microbial., 46, 565-601 (1992). 7. Suemori, A., Kurane, R., and Tomizuka, N.: Purification and properties of 3 types of monohydroxybenzoate oxygenases from Rhodococcus erythropolis S-l. Biosci. Biotech. Biochem., 57, 1487-1491 (1993). 8. Kurane, R., Ara, K., Nakamura, I., and Fukuoka, T.: Protocatechuate 3,4-dioxygenase from Nocardia erythropolis. Agic. Biol. Chem., 48, 2105-2111 (1984). 9. Suemori, A., Kuraue, R., and Tomizuka, N.: Purification and properties of gentisate 1,2_dioxygenase from Rhodococcus erythropolis S-l. Biosci. Biotech. Biochem., 57, 1781-1783 (1993). 10. Suemori, A., Kurane, R., and Tomizuka, N.: Purification and properties of phthalate oxygenase from Rhodococcus eryrhropolis S-l. Biosci. Biotech. Biochem., 57, 1482-1486 (1993). 11. Suemori, A., Nakajima, K., Kurane, R., and Yakamura, Y.: Degradation of aromatic amino acids by Rhodococcus erythropolis. Lett. Appl. Microbial., 21, 55-59 (1995). 12. Suemori, A., Nakajima, K., Kurane, R., and Nakamura, Y.: Degradative pathways of L-phenylalanine and L-tyrosine by Rhodococcus ervthrooolis. Reo. NIBH.. 3(2). 33-36 (1995). 13. Kurane, R., S&ki; T., Taiahara, l!., ‘aid Komagata; K.: Identification of phthalate ester-assimilating bacteria. Agric. Biol. Chem., 41, 1031-1038 (1977). 14. Suemori, A., Nakajima, K., Kurane, R., and Nakamura, Y.: Production of 3,4_dihydroxyphthalate by membrane-bound two-enzyme system from Rhodococcus erythropolis. Appl. Microbial. Biotech., 43, 470-472 (1995). 15. SuemorI, A., Nakajima, K., Kurane, R., and Nakamura, Y.: m- and p-Hydroxybenzoate degradative pathways in %odococcus erythropolis. FEMS Microbial. Lett., 125, 31-36 (1995). 16. Crawford, R. L. and Frick, T. D.: Rapid spectrophotometric differentiation between glutathione-dependent and glutathioneindependent gentisate and homogentisate pathways. Appl. Environ. Microbial., 34, 170-174 (1977). 17. Adachi, K. and Takeda, Y.: Homogentisate 1,2-dioxygenase. Meth. Enzymol., 17A, 638-642 (1970). 18. Crawford, R. L., Hutton, S. W., and Chapman, E. J.: Purification and properties of gentisate 1,2-dioxygenase from Moraxella osloensis. J. Bacterial., 121. 794-799 (1975). 19. Fujisawa, H., Uyeda, M., Kojima, Y., Nozaki, M., and Hay&hi, 0.: Protocatechuate 3,4-dioxygenase, II. Electron spin resonance and spectral studies on interaction of substrates and enzyme. J. Biol. Chem., 247, 4414-4421 (1972). 20. Crawford, R. L.: Degradation of homogentisate by strains of Bacillus and Moraxella. Can. J. Microbial., 22, 276-280 (1975). 21. Hagedorn, S. R., Bradley, G., and Chapman, P. J.: Glutathione-independent isomerization of maleylpyruvate by Bacillus megaterium and other gram-positive bacteria. J. Bacterial., 163, 640-647 (1985).
Purification and Characterization of o-Hydroxyphenylacetate SHydroxylase, m-Hydroxyphenylacetate 6-Hydroxylase and p-Hydroxyphenylacetate 1-Hydroxylase from Rhodococcus erythropolis AK10 SUEMORI,*
KENJI NAKAJIMA,
RYUICHIRO
National Institute of Bioscience and Human-Technology
KURANE,
AND YOSHIHIRO
NAKAMURA
(NIBH), I-i Higashi, Tsukuba, Ibaraki 305, Japan
Received 27 July 1995/Accepted 13 November 1995 The gram-positive Rhodococcus erythropolis strain Sl was found to utilize o-, m-, and p-hydroxyphenylacetic acids as sole carbon sources. Each isomer of monohydroxyphenylacetate was degraded via the homogentisate pathway, which contained a reduced glutathione independent-isomerase. Three monohydroxyphenylacetate monooxygenases, o-hydroxyphenylacetate 5-hydroxylase, m-hydroxyphenylacetate B-hydroxylase, and p-hydroxyphenylacetate 1-hydroxylase, were purified to homogeneity from strain Sl. Each enzyme was a 45kDa monomeric NADH-dependent monooxygenase containing FAD, and all three appeared to belong to the p-hydroxybenzoate hydroxylase-class as regards the flavin-containing aromatic compound monooxygenase family. However, the three enzymes differed greatly in terms of substrate specificity. [Key words: Rhodococcus erythropolis, o-hydroxyphenylacetate hydroxylase, p-hydroxyphenylacetate I-hydroxylase]
6-
Sigma (USA). Sodium salicylate, sodium m-hydroxybenzoate, sodium p-hydroxybenzoate, gentisic acid, and protocatechuic acid were purchased from Wako Pure Chemical Company (Osaka). Bacterium and media R. erythropolis strain Sl was isolated from soil with an enrichment method using a phthalate ester as sole carbon source (13). YMG medium consisted of 4 g of yeast extract (Difco), 10 g of malt extract (Difco), and 4 g of glucose in 1 1 of distilled water, pH 7.3. The minimal medium composition was the same as described previously (14). Strain Sl was preincubated in YMG medium with shaking at 30°C for 1 d. One ml of YMG-preculture was inoculated into 1OOml of minimal medium at O.l-O.3% monohydroxyphenylacetate and incubated with shaking at 30°C for 16 h. Cells in the logarithmic growth period were harvested by centrifugation and then washed twice in distilled water. They were then either used immediately in an oxygen uptake experiment or stored at -80°C until use for enzyme purification. Oxygen uptake The assay system for oxygen uptake by intact cells grown on aromatic compounds contained a suitable amount of washed cells and 2 mM substrate in a final volume of 2 ml of 10 mM sodium phosphate buffer, pH 7.1 (10). The oxygen consumption rate (OCR) was monitored using a biological oxygen monitor (YSI model 5300). As the control, the assay system, omitting substrate, was measured and OCR compared with the aromatic compound was defined by reducing the OCR of the substrate-omitted system from that of the complete assay system. Homogentisate pathway For characterization of the homogentisate pathway, a crude enzyme solution was prepared as follows. Frozen cells grown on 0.3% MHPA were suspended in a 10mM sodium phosphate buffer, pH 7.1, containing 10% (v/v) glycerol and disrupted by sonication for 18 min. The mixture was centrifuged (2O,OOOxg, 60min) and ammonium sulfate was added to the supernatant to 30% saturation. The crude
Many microorganisms have been shown to degrade aromatic compounds in the environment, but the mechanism of degradation of aromatic compounds by gram-positive bacteria and actinomycetes is not yet fully understood. In addition, the metabolism of phenylacetates and monohydroxyphenylacetates by microorganisms has been studied extensively (l-6), while little attention has been given to the functions of monohydroxyphenylacetate hydroxylases belonging to the flavin-containing aromatic compound monooxygenase family (4-6). The gram-positive Rhodococcus erythropolis strain Sl is capable of assimilating not only various aromatic carboxylic acids such as monohydroxybenzoates (7), dihydroxybenzoates (8, 9), and phthalate (10) but also aromatic amino acids (11). L-Tyrosine may also be degraded via formation of p-hydroxyphenylacetate and homogentisate by strain S 1 (12). Our attention has thus been directed to the degradation of monohydroxyphenylacetates using strain Sl. The degradation of o-, m-, and p-hydroxyphenylacetates using strain St and characterization of the three monohydroxyphenylacetatebreakdown pathways involving the homogentisate pathway are discussed in this report. Here, we also report on the purification and some of the properties of three monohydroxyphenylacetate monooxygenases, o-hydroxyphenylacetate 5-hydroxylase (OHPASH), m-hydroxyphenylacetate 6-hydroxylase (MHPA6H), and p-hydroxyphenylacetate 1-hydroxylase (PHPAlH), from strain Sl. MATERIALS
S-hydroxylase, m-hydroxyphenylacetate
AND METHODS
Materials o-Hydroxyphenylacetic acid (OHPA), mhydroxyphenylacetic acid (MHPA), p-hydroxyphenylacetic acid (PHPA), homogentisic acid (i.e. 2,5dihydroxyphenylacetic acid), and homoprotocatechuic acid (i.e. 3,4-dihydroxyphenylacetic acid) were obtained from * Corresponding author. 133
134
J. FERMENT. BIOENG.,
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extract was applied to a Butyl-Toyopearl 650M column equilibrated with the buffer 30% saturated with ammonium sulfate. After thoroughly washing the column with the same buffer to remove various elements such as NAD(P)H, the enzymes were eluted with the buffer without any ammonium sulfate. The major protein fraction was used for the enzyme assay and characterization of the homogentisate pathway. An enzyme reaction system containing 0.2mg of protein and 0.05 mM homogentisate in a final volume of 2 ml of 50mM HEPES/NaOH buffer, pH 8.0, at 24°C was used (15). The reaction was monitored spectrophotometrically based on the time course of absorbance at between 330 and 345 nm. For examination of the effects of reduced glutathione (GSH) or N-ethylmaleimide (NEM), GSH or NEM added to the reaction mixture to a final concentration of 25 mM (16). Enzyme assay The assay system for the monohydroxyphenylacetate monooxygenase activity contained a suitable amount of enzyme, 0.1 mM NADH, and 0.5 mM OHPA, MHPA, or PHPA in a final volume of 2 ml of 10 mM HEPES/NaOH buffer, pH 8.0. The enzyme activity was measured spectrophotometrically by measuring the decrease in the absorbance at 340 nm with oxidation of NADH. One unit of activity was defined as the amount of the enzyme that catalyzed the oxidation of NADH at a rate of 1 ,nmol per min at 30°C. Assay systems for the activity of homogentisate 1,2-dioxygenase (17), gentisate 1,Zdioxygenase (18), or protocatechuate 3,4-dioxygenase (19) each contained a suitable amount of enzyme, 0.5 mM homogentisate, gentisate, or protocatechuate, and 0.05 mM Fez+ in a final volume of 2 ml of 1OmM TAPS/NaOH buffer, pH 8.5. Enzyme activity was measured spectrophotometrically based on the increase in the absorbance at 330, 334, or 340nm, determined by the change in the amounts of maleylacetoacetate, maleylpyruvate, and P-carboxy-ciscis-muconate. One unit of activity was defined as the amount of enzyme catalyzing enzyme production in each case at a rate of 1 /*mol per min at 30°C. Purification and characterization of enzymes OHPASH, MHPA6H, and PHPAlH were purified from cell-free extracts of OHPA-, MHPA-, and PHPA-grown cells of strain Sl, respectively, as follows. All steps were carried out at below 5°C. The buffer used in all steps consisted of 10% (v/v) glycerol in a 10 mM sodium phosphate buffer, pH 7.0. Protein content was calculated from the absorbance of enzyme solutions at 260 and 280 nm. Step I. Crude extract Frozen cells (approximately 10 g) were suspended in a buffer and disrupted by sonication for 15 min. The turbid mixture was centrifuged (2O,OOOxg, 60min), and the resultant supernatant was used as the crude extract. Ammonium sulfate was added TABLE 1.
to the crude extract to 30% saturation. Step 2. Chromatography on Butyl- Toyopearl6SOM The enzyme solution was applied to a Butyl-Toyopearl 650M column equilibrated with a buffer 30% saturated with ammonium sulfate. After the column was washed with the same buffer, the enzyme was eluted with buffer with a linear 30-10% ammonium sulfate saturation gradient at 1.5 ml per min. The active fractions were collected and ammonium sulfate was added to the enzyme solution to obtain a saturation of 70%. The solution was left for 1 d. After centrifugation at 20,000~ g for lOmin, the precipitate was dissolved minimum volume of buffer. Step 3. Gel filtration on Toyopearl HW-55F The enzyme solution was applied to a Toyopearl HW-55F column equilibrated with buffer, and eluted with the same buffer at 0.38 ml per min. The active fractions were combined. Step 4. Chromatography on DEAE-Toyopearl65OS The enzyme solution was applied to a DEAE-Toyopearl 650s column equilibrated with the buffer. After the column was washed with the same buffer, the enzyme was eluted with the buffer with a linear O-O.3 M NaCl gradient at 0.38 ml per min. The active fractions were pooled and used for analysis of the properties of the purified enzyme. The purity of the enzyme at each purification step was assayed by native PAGE. This was carried out using a linear 5-20% polyacrylamide gel gradient in 0.025 M Tris and 0.192 M glycine buffers. The gel was stained with Coomassie brilliant blue G-250. The molecular weight of each enzyme was estimated by gel filtration using a Toyopearl HWSSF column. The column was equilibrated with a 10mM sodium phosphate buffer, pH 7.0, with 0.1 M NaCl. As reference proteins, ,%galactosidase (M, 540,000), catalase (Mr 244,000), bovine serum albumin dimer (Mr 130,000), peroxidase (Mr 44,000), and cytochrome c (M, 13,000) were used. The molecular weight of each enzyme was also estimated using SDS-PAGE with a linear S-20% polyacrylamide gel gradient in a 0.025 M Tris and 0.192 M glycine buffer with 0.1% SDS. Myosin (Mr 212,000), az-macrogloblin (Mr 170,000), $-galactosidase (Mr 116,000), transferrin (IV& 76,000), and glutamic dehydrogenase (Mr 53,000) were used as the standard marker proteins. The fluorescence of the enzyme was measured using the enzyme solution dialyzed completely against the 10 mM sodium phosphate buffer, pH 7.0. RESULTS Growth on three monohydroxyphenylacetates Strain Sl grew on OHPA, MHPA, or PHPA as sole carbon source (data not shown). Doubling times on the sub-
Oxygen consumption rate with monohydroxyphenylacetates and dihydroxyphenylacetates by intact cells of R. erythropolis Sl grown on monohydroxyphenylacetates, homogentisate, or YMG medium Oxygen consumption rate (nmol/rnin/ml/0.D.660)
Substrate
OHPA-grown
OHPA MHPA PHPA Homogentisate Homoprotocatechuate
81 9 10 96 4
MHPA-grown 8 71 12 138 3
PHPA-grown 3 11 74 120 6
Homogentisate-grown 9 13 16 173 7
YMG medium-grown 4 6 7 9
1
OHPA, MHPA, and PHPA-grown 31 36 40 106 2
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TABLE 2. Assay of enzyme activity in crude extracts prepared from cells of R. eryfhropofis Sl grown on OHPA, MHPA, or PHPA Enzyme activity assayed
Enzyme activity (Wmg-protein) OHPA-grown MHPA-grown PHPA-grown
OHPASH 2.8 MHPABH 0.3 PHPAlH 0.2 SALSH
0.4 3.1 0.2
0.1 0.3 4.0
strates were essentially the same, though with OHPA or PHPA, a lag time for 12-24 h was observed. Also, strain Sl grew on homogentisate and homoprotocatechuate as sole carbon sources. Induction of enzymes OCR compared against the monohydroxyphenylacetates and dihydroxyphenylacetates by intact cells grown on 0.2% (w/v) monohydroxyphenylacetate, 0.2% (w/v) dihydroxyphenylacetate, or YMG medium was measured (Table 1). Cells that were grown on OHPA demonstrated full oxygen uptake with OHPA and homogentisate. When grown with MHPA, the full oxygen uptake of MHPA and homogentisate could be observed whereas cells grown on PHPA showed full oxygen uptake with PHPA and homogentisate. Cells grown on OHPA, MHPA, and PHPA failed to have any oxygen uptake with homoprotocatechuate. Those grown on homogentisate showed almost only homogentisate after being oxidized. Cells grown in YMG medium failed to oxidize OHPA, MHPA, PHPA, or homogentisate. All three isomers thus appear to undergo hydroxylation to homogentisate due to strain Sl and the oxidation of each monohydroxyphenylacetate to be induci-
OF AROMATIC COMPOUND MONOOXYGENASES
135
ble by use of an appropriate monohydroxyphenylacetate as the growth substrate. On the other hand, after the cells were incubated in minimal medium containing all three isomers, the oxygen uptake with all three isomers by intact cells was observed (Table 1). The crude extract from cells grown on OHPA, MHPA, or PHPA was prepared and examined with respect to various enzyme activities (Table 2). Crude extracts from cells grown on OHPA showed NADH-oxidizing activity only in the presence of OHPA. Crude extracts from MHPA- or PHPA-grown cells exhibited enzyme activity only in the presence of MHPA or PHPA, respectively. It thus appears that each of the three enzymes may be involved in hydroxylation of the three monohydroxyphenylacetates, respectively. The enzymes were designated as o-hydroxyphenylacetate 5-hydroxylase, OHPASH, hydroxylating OHPA to homogentisate; m-hydroxyphenylacetate 6-hydroxylase, MHPA6H, hydroxylating MHPA to homogentisate; and p-hydroxyphenylacetate 1-hydroxylase, PHPAlH, catalyzing the hydroxylation and decarboxylation of PHPA to homogentisate (Fig. 1). All hydroxylation reactions shown by crude extracts a strict preference for NADH. The crude extracts from cells grown on the three monohydroxyphenylacetate isomers expressed full homogentisate 1,2dioxygenase activity. There was hardly any gentisate 1,2dioxygenase or protocatechuate 3,4-dioxygenase activity. Thus, we concluded that in the degradation of monohydroxyphenylacetates, only the homogentisate pathway was induced and used. GSH-independent bomogentisate pathway In the homogentisate pathway characterization, a shift in the absorbance maximum from 330 to 345 nm could be observed, indicating conversion of maleylacetoacetate to fumarylacetoacetate as a result of isomerase. A decrease in absorbance at 330 nm was not promoted by GSH, nor was it inhibited by NEM in the presence of GSH (16).
COOH
0
OH
&
(1)
\
cFHydroxyphenylacetate
mHydroxyphenylacetate
Homogentisate
Maleylacetoacetate
Fumarylacetoacetate
COOH
4 0
/
(3)
pHydroxyphenylacetate FIG. 1. Degradative pathways of OHPA, MHPA and PHPA by R. erythropolis Sl. (1). o-Hydroxyphenylacetate 5-hydroxylase; (2), mhydroxyphenylacetate 6-hydroxylase; (3), p-hydroxyphenylacetate I-hydroxylase; (4), homogentisate 1,2-dioxygenase; (5), GSHindependent maleylacetoacetate isomerase.
136
J. FERMENT.BIOENG.,
SUEMORI ET AL. TABLE 3.
Summary of purification procedures of OHPASH, MHPA6H, and PHPAlH OHPASH
Steps
Specific activity (U/m& Crude extract 12 Butyl-Toyopearl 192 Gel filtration 576 DEAE-Toyopearl 2100
Yield (%) 100 78 49 21
MHPA6H Purification (fold) 1 16 48 175
Specific activity (v/m& 16 176 1104 2544
Similar findings were observed for the pathway of Bacillus sp. (20). The relationship of the time course of 330 and 345 nm was virtually the same as that for the pathways involving the GSH-independent maleylacetoacetate isomerase (21). Accordingly, the following ring-cleavage of homogentisate by homogentisate 1,Zdioxygenase in strain Sl, maleylacetoacetate may be converted to fumarylacetoacetate due to GSH-independent maleylacetoacetate isomerase activity (Fig. 1). Homogentisate was also found to be degraded via the GSH-independent homogentisate pathway in strain Sl, which is in good agreement with findings on the GSH-independent maleylacetoacetate isomerases in some gram-positive microorganisms (19-21). Strain Sl was previously shown to possess the GSH-independent gentisate pathway (15). All the results of the present study are consistent with the fact that gram-positive bacteria possess a GSHindependent (homo)gentisate pathway (21). Puritication and characterization of three enzymes The purification procedures for the three enzymes, OHPASH, MHPA6H, and PHPAlH, are summarized in Table 3. Each of the three purified enzymes gave a single protein band on SDS-PAGE (data not shown). OHPASH, MHPA6H, and PHPAlH were purified approximately 175, 159, and 194-fold with 21, 14, and 19% activity yield, respectively. The molecular weights of OHPASH, MHPA6H, and PHPAlH were estimated to be 49, 50, and 49 kDa by gel filtration but were all estimated to be 45 kDa by SDS-PAGE. Each native enzyme thus appears to be a monomer. As fluorescence examination, after emission at 520 nm, the excitation spectrum showed their maxima at 375 nm and 454 nm. When excited at 450 nm, an emission maximum was observed at 520nm. Each spectrum was in good agreement with that of FAD. The three enzymes were thus concluded to be flavin-containing aromatic compound monooxygenases. The activity of each enzyme was examined using each TABLE 4.
Substrate specificities of OHPASH, MHPA6H, and PHPAlH from R. erythropolis Sl
Substrate o-Hydroxyphenylacetate m-Hydroxyphenylacetate p-Hydroxyphenylacetate Sahcylate m-Hydroxybenzoate p-Hydroxybenzoate o-Hydroxyphenoxyacetate m-Hydroxyphenoxyacetate p-Hydroxyphenoxyacetate p-Hydroxypropionate n-(BenzylalcohoBacetate NT, Not tested.
Relative activity (%) OHPASH MHPA6H PHPAlH 100 <5 <5 6 NT NT 26 NT NT NT NT
from R. erythropolis Sl
<5
<5
100 <5 NT <5 NT NT 19 NT NT NT
<5 100 NT NT <5 NT NT 32 28 <5
Yield (%) 100 86 30 14
PHPAlH Purification (fold) 1 11 69 159
Specific activity (U/mg) 14 280 1316 2716
Yield (%) 100 73 24 19
Purification (fold) 1 20 94 194
of several aromatic compounds as a substrate (Table 4). OHPASH oxidized mainly OHPA and to some extent ohydroxyphenoxyacetate. MHPA6H showed the strongest activity toward MHPA and some toward m-hydroxyphenoxyacetate. PHPAlH reacted with PHPA and to a slight degree with p-hydroxyphenoxyacetate and phydroxyphenylpropionate. Thus, the substrate specificities of the enzymes varied greatly. On the other hand, all enzymes were specific for NADH; i.e., they showed no activity toward NADPH. DISCUSSION These results support the view that all three isomers of monohydroxyphenylacetate are converted by hydroxylation to homogentisate by strain Sl, which produces these three enzymes that clearly differ from monohydroxybenzoate hydroxylases such as salicylate 5-hydroxylase (SALSH), m-hydroxybenzoate 6-hydroxylase (MHB6H), and p-hydroxybenzoate 3-hydroxylase (PHB3H) (7). All three monohydroxyphenylacetate hydroxylases from strain Sl were shown to be 45-kDa monomers, which is in good agreement with the subunit size of PHPAlH from Pseudomonas acidovorans (3) and 4-hydroxyphenylacetate 3-hydroxylase (PHPA3H) from Pseudommonas putida (4), as well as SALSH, MHB6H, and PHB3H from strain Sl (7). These data strongly suggest that the three monohydroxyphenylacetate hydroxylases from strain Sl belong to the p-hydroxybenzoate hydroxylase class of enzymes which have a 45-kDa subunit (6). Furthermore, all three monohydroxyphenylacetate hydroxylases from strain Sl were shown to have strong preference for NADH as did the three monohydroxybenzoate hydroxylases from the same strain (7). The co-substrate specificities of purified monohydroxyphenylacetate hydroxylases differ somewhat. For instance, MHPA6H from Flavobacterium sp. JS-7 (5) and PHPAlH from P. acidovorans (3) use NADH and NADPH with essentially the same efficiency. However, PHPAlH from Klebsiella pneumoniae (2) and PHPA3H from P. putida (4) display strong NADH preference. It is quite interesting that one bacterial strain produces various inducible flavin-containing aromatic compound monooxygenases such as OHPASH, MHPABH, PHPAlH, SALSH, MHB6H, and PHB3H having the same subunit sizes but narrow substrate specificity. Most flavin-containing aromatic compound monooxygenases catalyze the incorporation a new hydroxyl group in ortho position onto an existing hydroxyl group (21); however, all three monohydroxyphenylacetate hydroxylases from strain Sl catalyze para hydroxylation, as do SALSH and MHB6H. In consideration of the mode of hydroxylation of monohydroxylated aromatic rings, OHPASH, MHPA6H, and PHPAlH appear functionally more similar to SALSH and MHB6H than PHB3H in strain S 1.
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VOL. 81, 1996
In consideration of the patterns of the inducement of oxidative activity for monohydroxyphenylacetates, for example, MHPA-grown cells of Flawbacterium sp. JS-7 could oxidize both MHPA and PHPA, while PHPAgrown cells showed a very low level of oxidative activity with MHPA (1). Washed cells of K. pneumoniae M5al grown on MHPA or PHPA oxidized MHPA and PHPA (2). These findings are somewhat at a variance with those on the effects of the growth substrate on the induction of MHPA6H or PHPAlH in strain Sl. The manner in which monohydroxyphenylacetate degradative pathways in microorganisms as well as monohydroxybenzoate degradative pathways are regulated and may not be the same in all cases (15). It is considered that m-hydroxybenzoate and p-hydroxybenzoate are catabolite repressors and may affect the level of the oxidative activity of salicylate in strain Sl, however, repression by other monohydroxyphenylacetate isomers was not observed in this study during the growth of strain Sl on monohydroxyphenylacetates. The three monohydroxyphenylacetate hydroxylases and three monohydroxybenzoate hydroxylases from strain Sl may be derived from a common ancestral protein/gene, and thus, their physicochemical and genetic properties should be carefully examined, REFERENCES I
2,
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van den Tweel, W. J. J., bits, J. P., and de Boot, J. A.M.: Catabolism of DL-a-phenylhydracrylic, phenylacetic and 3- and 4-hydroxyphenylacetic acid via homogentisic acid in a Fluvobacterium sp.-Arch. Microbial., 149, 207-213 (1988). Martin. M.. Gibello. A., FemandCz. J.. Ferrer. E.. and GarridoPertieria, A.: Catadolis’m of 3- and 4-hydrox$ph;nylacetic acid by Klebsiella pneumoniae. J. Gen. Microbial., 132, 621-628 (1991). Hareland, W. A., Crawford, R. L., Chapman, P. J., and DagIey, S.: Metabolic function and properties of 4-hydroxyphenylacetic acid 1-hydroxylase from Pseudomonas acidovorans. J. Bacterial.. 121. 272-285 (1975). Raju, S. G., Barn&, A. V., and iaidyanathan, C. S.: Purification and properties of 4-hydroxyphenylacetic acid 3-hydroxylase from Pseudomonas putidu. Biochem. Biophys. Res. Commun., 154, 537-543 (1988). van Berkel, W. J. H. and van den Tweel, W. J. J.: Purification and characterization of 3-hydroxyphenylacetate 6-hydroxylase: a novel FAD-dependent monooxygenase from a Flavobacteriurn species. Eur. J. Biochem., 201, 585-592 (1991). Harayama, S., Kok, M., and Neidle, E. L.: Functional and evolutionary relationships among diverse oxygenases. Annu.
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Rev. Microbial., 46, 565-601 (1992). 7. Suemori, A., Kurane, R., and Tomizuka, N.: Purification and properties of 3 types of monohydroxybenzoate oxygenases from Rhodococcus erythropolis S-l. Biosci. Biotech. Biochem., 57, 1487-1491 (1993). 8. Kurane, R., Ara, K., Nakamura, I., and Fukuoka, T.: Protocatechuate 3,4-dioxygenase from Nocardia erythropolis. Agic. Biol. Chem., 48, 2105-2111 (1984). 9. Suemori, A., Kuraue, R., and Tomizuka, N.: Purification and properties of gentisate 1,2_dioxygenase from Rhodococcus erythropolis S-l. Biosci. Biotech. Biochem., 57, 1781-1783 (1993). 10. Suemori, A., Kurane, R., and Tomizuka, N.: Purification and properties of phthalate oxygenase from Rhodococcus eryrhropolis S-l. Biosci. Biotech. Biochem., 57, 1482-1486 (1993). 11. Suemori, A., Nakajima, K., Kurane, R., and Yakamura, Y.: Degradation of aromatic amino acids by Rhodococcus erythropolis. Lett. Appl. Microbial., 21, 55-59 (1995). 12. Suemori, A., Nakajima, K., Kurane, R., and Nakamura, Y.: Degradative pathways of L-phenylalanine and L-tyrosine by Rhodococcus ervthrooolis. Reo. NIBH.. 3(2). 33-36 (1995). 13. Kurane, R., S&ki; T., Taiahara, l!., ‘aid Komagata; K.: Identification of phthalate ester-assimilating bacteria. Agric. Biol. Chem., 41, 1031-1038 (1977). 14. Suemori, A., Nakajima, K., Kurane, R., and Nakamura, Y.: Production of 3,4_dihydroxyphthalate by membrane-bound two-enzyme system from Rhodococcus erythropolis. Appl. Microbial. Biotech., 43, 470-472 (1995). 15. SuemorI, A., Nakajima, K., Kurane, R., and Nakamura, Y.: m- and p-Hydroxybenzoate degradative pathways in %odococcus erythropolis. FEMS Microbial. Lett., 125, 31-36 (1995). 16. Crawford, R. L. and Frick, T. D.: Rapid spectrophotometric differentiation between glutathione-dependent and glutathioneindependent gentisate and homogentisate pathways. Appl. Environ. Microbial., 34, 170-174 (1977). 17. Adachi, K. and Takeda, Y.: Homogentisate 1,2-dioxygenase. Meth. Enzymol., 17A, 638-642 (1970). 18. Crawford, R. L., Hutton, S. W., and Chapman, E. J.: Purification and properties of gentisate 1,2-dioxygenase from Moraxella osloensis. J. Bacterial., 121. 794-799 (1975). 19. Fujisawa, H., Uyeda, M., Kojima, Y., Nozaki, M., and Hay&hi, 0.: Protocatechuate 3,4-dioxygenase, II. Electron spin resonance and spectral studies on interaction of substrates and enzyme. J. Biol. Chem., 247, 4414-4421 (1972). 20. Crawford, R. L.: Degradation of homogentisate by strains of Bacillus and Moraxella. Can. J. Microbial., 22, 276-280 (1975). 21. Hagedorn, S. R., Bradley, G., and Chapman, P. J.: Glutathione-independent isomerization of maleylpyruvate by Bacillus megaterium and other gram-positive bacteria. J. Bacterial., 163, 640-647 (1985).