Purification and characterization of nicotinic acid dehydrogenase from Pseudomonas fluorescens TN5

J o t r ~ A t OF FERMENTATIONAND BIOENGINEERING Vo1. 78, No. 1, 19-26. 1994

Purification and Characterization of Nicotinic Acid Dehydrogenase from Pseudomonas fluorescens TN5 BYUNGSERK H U R H , TSUNEO YAMANE, AND TORU NAGASAWA*

Department of Food Science and Technology, Faculty of Agriculture, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464, Japan Received 24 January 1994/Accepted 6 April 1994 Membrane-bound nicotinic acid dehydrogenase, an enzyme that catalyzes the formation of 6-hydroxynicotinic acid from nicotinic acid, was solubilized with Triton X-100, and then purified 126-fold with an 11.1% overall recovery from nicotinic acid-induced cells of Pseudomonasfluorescens TN5. The purified enzyme appeared to be homogeneous from analysis by polyacrylamide gel electrophoresis. The enzyme had a molecular mass of approximately 80 kDal and consisted of one subunit. Some electron acceptors, such as phenazine methosulfate, K3Fe(CN)6 and nitro blue tetrazolium, acted as electron acceptors. The purified enzyme catalyzed the hydroxylation of nicotinic acid to 6-hydroxynicotinic acid at a rate of 672 pmol min -1 mg -1 of protein at 35°C. It also catalyzed the hydroxylation of pyrazinecarboxylic acid, 3-pyridinesulfonic acid, and 3cyanopyridine. The purified enzyme exhibited an optimum pH of 8.3, and was sensitive to thiol reagents such as HgCI2 and p-ehioromercuribenzoate. A reduction in the amount of cytochrome c-like component in the respiratory particles was observed during the hydroxylation reaction of nicotinic acid. Thus, nicotinic acid dehydrogenase appeared to be linked to the cytochrome respiratory chain in the cells of P. fluorescens TN5.

Microbial hydroxylation reactions of aromatic and heterocyclic compounds are characterized by positionspecificity and high efficiency. Thus, they have been applied for the production of useful compounds in the fields of medicine and chemical engineering (1-11). However, in contrast with the hydroxylation of benzene and its derivatives, few reports have dealt with the hydroxylation of heterocyclic compounds (12-16). Recently, new insecticides containing the 3-chloropyridylmethyl group as a versatile building block have been developed (17-22), and imidachloprid is one of the most promising insecticides among them. 6-Hydroxynicotinic acid is the first intermediate of the bacterial degradation pathway of nicotinic acid (23-26). It may be useful as a starting material for the synthesis of these chloronicotinyl insecticides. Recently, we surveyed the conversion activity of nicotinic acid into 6-hydroxynicotinic acid by various bacteria. The resting cells of Serratia marcescens IFO 12648, Comamonas acidovorans and Pseudomonas fluorescens TN5 were found to accumulate an enormous amount of 6-hydroxynicotinic acid from nicotinic acid with a high conversion yield (27, 28). The enzyme responsible for the position-6-specific hydroxylation of nicotinic acid by P. fluorescens TN5 was found to be localized in the membrane-bound fraction (29, 30). On the other hand, the enzymes from Bacillus niacini (31-34) and Clostridium barkeri (35-38) were soluble. In the present study, we attempted to purify and characterize the membrane-bound nicotinic acid hydroxylation enzyme of P. fluorescens TN5. Then a discussion was made in comparison with soluble hydroxylation enzymes from B. niacini and C. barkeri.

MATERIALS AND METHODS Microorganism and cultivation conditions P. fluorescens TN5 was collected from an agar slant and inoculated into a subculture which was shaken at 28°C for 24h in a test tube (17 × 165 mm) containing a 3-ml medium consisting of 5g Polypepton (Nippon Seiyaku, Tokyo), 0.5 g yeast extract (Oriental Yeast, Tokyo), 5 g meat extract (Mikuni, Tokyo), and 2 g NaC1 per 100 ml of tap water (pH 7.0). The subculture (6 ml) was inoculated into a 3-/shaking flask containing 300 ml of a medium comprised of 1 g nicotinic acid, 1 g malic acid, 1 g meat extract, 0.1 of yeast extract, 0.1 g KEHPO4, 0.05 g M g S O 4. 7H20, and 1 ml metal solution per 100 ml of tap water (pH 7.0). It was then incubated for 28 h at 28°C with rotary shaking (120rpm). The metal solution consisted of 40 mg CaCI:.2H20, 50 mg HBO3, 4 mg CuSO4. 5H20, 10mg K1, 20mg FeSO4.7H20, 40rag MnSO4. 7H20, 40mg ZnSO4.7HEO, 20mg HEMOO4.2HEO, and 2 ml conc. HC1 per 100 ml of distilled water. The cells harvested by centrifugation at 7,000 × g at 4°C were suspended in 0.1 M potassium phosphate buffer (pH 7.0). By repeating the centrifugation and suspension process, the cells were thoroughly washed. Enzyme assay Enzyme assay was carried out by measuring spectrometrically the reduction of electron acceptor in accordance with the formation of 6-hydroxynicotinic acid or by measuring 6-hydroxynicotinic acid formed by high performance liquid chromatography (HPLC). The spectrophotometric assay was performed according to Nagel and Andreesen's method (34) using phenazine methosulfate (PMS) coupled with dichloroindophenol (DCPIP) as an electron acceptor. The reaction was carried out at 35°C in a cuvette containing 2.7 ml of the reaction mixture comprising 250/zmol of potassium phosphate buffer (pH 7.0), 30/~mol of nicotinic acid, 0.15ftmol of DCPIP, 0.30/~mol of PMS, and an appropriate amount of the enzyme solution. The reduction

* Corresponding author. 19

20

HURH ET AL.

of DCPIP was measured at 610nm with a Jasco 660 spectrophotometer (Jasco, Tokyo). In the determination of the amount of 6-hydroxynicotinic acid formed by HPLC, the reaction was carried out in a 50-ml beaker (3.5 x 6.0 cm) containing a 2-ml reaction mixture. The reaction mixture contained 160pmol of potassium phosphate buffer (pH 7.0), 200pmol of nicotinic acid, 100pmol of PMS, and an appropriate amount of the enzyme solution. The reaction was carried out at 35°C for 10 min with reciprocal shaking (160 strokes per min at 3.5 cm amplitude) and stopped by adding 1 ml of 0.2 N HC1. Determination of 6-hydroxynicotinic acid was carried out by HPLC equipped with an M&S pack C-18 column (reverse phase, 46 × 150mm; M&S Instruments) at a flow rate of 1.0ml/min, using the following solvent system: 10mM KH2PO4-H3PO4 (pH 2.5)/acetonitrile (98 : 2, by volume). The eluate was monitored at 210 nm with a Uvidec-100-V (Jasco). One unit of enzyme activity was defined as the amount of enzyme needed to catalyze the formation of 1 pmol of 6-hydroxynicotinic acid per min under the conditions described above. The presence of protein was determined by the Coomassie brilliant blue G-250 dye-binding method of Bradford (39) using the dye reagent supplied by Bio-Rad (USA). Enzyme purification All purification steps were performed at 0-4°C. Throughout the purification, potassium phosphate buffer (pH7.0) containing 20mM nicotinic acid was used unless otherwise specified. Step 1. Solubilization of the enzyme Washed cells (approximately 90 g dry weight) were suspended in 800ml of the 0.1 M buffer, disrupted for 10min by ultrasonic oscillation (19kHz, Insonator model 201M; Kubota), and then centrifuged at 100,000x g for 60 min at 4°C with an ultracentrifuge (Hitachi 55P-7). The precipitates were then suspended in 0.1 M the buffer containing 0.3% (w/v) Triton X-100. After stirring overnight at 0°C with a magnetic stirrer, the suspension was centrifuged again at 100,000xg for 60min at 4°C. The reddish-colored supernatant obtained exhibited hydroxylation activity, and the enzyme solution was designated as the solubilized enzyme.

Step 2. First DEAE-Sephacel column chromatography The solubilized enzyme solution was dialyzed against the 0.01 M buffer containing 0.3% (w/v) Triton X-100. The dialyzed enzyme solution was applied to a DEAE-Sephacel column (3.3 x 2 8 c m ) equilibrated with the 0.01 M buffer containing 0.3% (w/v) Triton X-100. After the column was washed with 500ml of the same buffer, the enzyme was eluted with the 0.1 M buffer containing 0.3% (w/v) Triton X-100. The active fractions (8.3ml/fraction) were combined and dialyzed against the 0.01 M buffer containing 0.3% (w/v) Triton X-100. Step 3. Second DEAE-Sephacel column chromatography The enzyme solution from step 2 was applied to a DEAE-Sephacel column (3.3 x 28cm) equilibrated with the 0.01M buffer containing 0.3% (w/v) Triton X-100. After washing the column with 500 ml of 0.01 M the buffer, the enzyme was eluted with a linear gradient of KC1 (0 to 0.4M, total volume 1,000 ml) at a flow rate of 40 ml/h. The active fractions (9.6 ml/fraction) were combined and ammonium sulfate was added to 20% saturation. Step 4. Phenyl Sepharose CL-4B column chromatography The enzyme solution from step 3 was ap-

J. F ~ N T . BIO~NO., plied to a phenyl Sepharose CL-4B column (1.7 × 28 cm) equilibrated with the 0.01 M buffer containing 20% saturated ammonium sulfate. After washing the column with 150ml of the 0.01 M buffer containing 20% saturated ammonium sulfate, followed by another washing with 150 ml of the 0.01 M buffer containing 5% saturated ammonium sulfate, the enzyme was eluted by the 0.01 M buffer. The active fractions (5.3 ml/fraction) were combined and ammonium sulfate was added to 20% saturation.

Step 5. First butyl Toyopearl column chromatography The enzyme solution from step 4 was applied to a butyl Toyopearl column (1.7×20cm) equilibrated with the 0.01 M buffer containing 20% saturated ammonium sulfate. After the column was washed in 100ml of the equilibration buffer, the enzyme was eluted by the 0.01 M buffer containing 10% saturated ammonium sulfate. The active fractions (4.7 ml/fraction) were combined and ammonium sulfate was added to 20% saturation. Step 6. Second butyl Toyopearl column chromatography The enzyme solution from step 5 was applied to a butyl Toyopearl column ( 1 . 7 x 2 0 c m ) equilibrated with the 0.01 M buffer containing 20% saturated ammonium sulfate. After successive washing of the column with 100ml of the equilibration buffer and 100 ml of 0.01 M potassium phosphate buffer containing 15% saturated ammonium sulfate, the enzyme was eluted by 0.01 M potassium phosphate buffer containing 12.5% saturated ammonium sulfate. The active fractions (5 ml/fraction) were then combined. Analytical method for nicotinic acid dehydrogenase SDS-PAGE was performed in 10% (w/v) polyacrylamide slab gels using the Tris/glycine buffer system (40). The molecular mass of the subunit of the enzyme was determined from the relative mobilities of standard proteins (a low-molecular-mass standard kit obtained from Pharmacia, Sweden). Native PAGE was performed in 10% (w/v) polyacrylamide slab gel according to the method of Laemmli (40) except that SDS and 2-mercaptoethanol were omitted. The gels were stained for protein with Coomassie brilliant blue R-250. Activity staining of the native PAGE was performed by incubating the gel for 20min in 4 0 m M potassium phosphate buffer (pH7.0) containing 20mM nicotinic acid, 0.5mM nitro blue tetrazolium and 1.0% (w/v) Triton X-100 (32). The color of nitro blue tetrazolium developed in accordance with the hydroxylation of nicotinic acid catalyzed by nicotinic acid dehydrogenase. To detect the presence of the heine compounds, staining for heme-associated peroxidase activity was performed according to the method of Tomas et al. (41). The enzyme sample was subjected to H P L C on a TSK G-3000SW column (0.75 × 60 cm, Toyo-Soda) at a flow rate of 0.7 ml/min with the elution of 0.1 M potassium phosphate buffer (pH 7.0) containing 0.2 M NaCI as the eluant at room temperature. The absorbance of the effluent was recorded at 280 nm. The molecular mass of the enzyme was then calculated from the relative retention times compared to standard proteins (products of Oriental Yeast Co. Ltd., Tokyo), glutamate dehydrogenase (290kDal), lactate dehydrogenase (140kDal), enolase (67 kDal), adenylate kinase (32 kDal), and cytochrome c (12.4 kDal). Preparation of respiratory particles Respiratory particles were prepared using a modification of the

VoL. 78, 1994

NICOTINIC ACID HYDROXYLATION

method described by Sweet and Peterson (42). Approximately 3 g of wet cells was suspended per 100ml of 20 mM potassium phosphate buffer (pH 7.4) at room temperature. EDTA was added to make a final concentration of 10mM, lysozyme (Seikagaku Kogyo Co. Ltd.) was added to make 0.2 mg/ml, and the suspension was stirred for 15 min to allow most of the cells to lyse. MgSO4 was added to make 20 raM, and the pH was maintained near neutral by the dropwise addition of 1 N KOH. Deoxyribonuclease I (Sigma, St. Louis, USA) was added to make 10 pg/ml, stirring was continued for an additional 15 min, and the suspension was then cooled to 5°C. The suspension was centrifuged for 10min at 1,000 z g in a Kubota 7820 centrifuge, after which the supernatant was carefully decanted and the pellet discarded. This procedure was repeated three times to insure the removal of unlysed cells and the largest membrane fragments, and the supernatant was then centrifuged at 30,000xg in a Kubota 7820 centrifuge at 4°C for 45 rain. The clear supernatant was decanted and discarded. The pellet was washed by resuspending with gentle hand homogenization in 0.1 M potassium phosphate buffer, pH7.4, containing 10mM EDTA, and the 30,000xg centrifugation step was repeated. The pellet was then resuspended by gentle homogenization in 1 to 2 volumes of the phosphate-EDTA buffer used for the washing step to give a final protein concentration of 20 to 30mg/ml. Approximately 3 g of wet ceils yielded about 2 ml of respiratory particles. RESULTS AND DISCUSSION Purification of nicotinic acid dehydrogenase Using the procedures described under Materials and Methods, the enzyme was purified 126-fold with a yield of 11.1°~ from the solubilized enzyme solution (Table 1). The purified enzyme migrated as a single species on SDSPAGE (Fig. IA), as judged by protein staining. On nondenaturing PAGE (Fig. 1B), the band of purified protein coincided with that of the nicotinic acid dehydrogenaseactivity staining. These results suggest that the enzyme preparation is homogeneous. This is the first time that microbial membrane-bound nicotinic acid dehydrogenase has been purified. The purified enzyme catalyzed the hydroxylation of nicotinic acid to 6-hydroxynicotinic acid at 672/~mol min -~ (mg protein) -1 under the standard reaction conditions. Our specific activity [672ffmol min -~ (mg protein) -1 at 35°C] was the highest among those reported for other nicotinic acid dehydrogenases: Bacillus niacini DSM 2923 enzyme, 2.2 at 30°C (34); Clostridium sp., 28.8 at 30°C (35); and enzyme partially purified from P, fluorescens KB1, 60 at 25°C (29). Molecular mass and subunit structure Gel-permea-

A 1

B 2

I

2

94.0 kDal 67.0

43.0

30.0

20.0 4" 14.4

4FIG. 1. SDS (A) and native (B) gel electrophoreses of nicotinic acid dehydrogenase. (A) The conditions for SDS-PAGE are given under Materials and Methods. Lane I, the purified enzyme (8 pg). Lane 2, marker proteins: phosphorylase b, 94 kDal; bovine serum albumin, 67 kDal; ovalbumin, 43 kDal; carbonic anhydrase, 30 kDal; soybean trypsine inhibitor, 20.0 kDal; ct-lactalbumin, 14.4kDal. (B) The conditions for native PAGE and activity staining are given under Materials and Methods. Lane 1, Coomassie brilliant blue R-250 staining; lane 2, activity staining. tion HPLC yielded only one absorption peak of protein. The molecular mass of the purified nicotinic acid dehydrogenase was determined to be 100 kDal by gel-permeation HPLC. When the enzyme treated with l°Jo SDS and 50 mM 2-mercaptoethanol was electrophoresed on a gel containing 0 . 1 ~ SDS, a single band was observed with protein dying. The molecular mass corresponding to the band was estimated to be 80 kDal based on its mobility relative to those of reference proteins. The enzyme probably consists of one subunit. The purified enzyme contained no substances showing any absorbance in the visible region of 350-750nm. Peaks other than that at 280 nm did not appear in the ultraviolet region of 250-350nm. Hunt (29) reported the occurrences of cytochromes c, cl, bl and flavoprotein components in the partially purified nicotinic acid dehydrogenase from P. fluorescens KB1, which were rapidly reduced by the addition of nicotinic acid. However, in contrast with his results, we could not detect any cytochromes or flavoprotein components in the highly purified nicotinic acid dehydrogenase itself. It has been reported that the molecular mass of B. niacini DSM 2923 soluble enzyme is 300 kDal and that the

TABLE 1. Purification of nicotinic acid dehydrogenasefrom P. fluorescens TN5 Step Solubilized enzyme DEAE Sephacel (lst) DEAE Sephacel (2nd) Phenyl Sepharose CL-4B Butyl Toyopearl (1st) Butyl Toyopearl (2nd)

Total protein (mg) 3510 595 183 23.1 10.4 3.08

Total activity (units) 18700 8990 6220 3390 2310 2070

The assay was carried out under the standard reaction conditions.

21

Specificactivity (units/mg) 5.33 15.1 34.0 147 222 672

Fold 1 2.83 6.38 27.6 41.7 126

Yield (%) 100 48.1 33.3 18.1 12.4 11.1

22

HURH ET AL.

J. FERM~r~T. BIOENG., TABLE 2.

Substrate specificity of nicotinic acid dehydrogenase

A

l

Km

(mM)

COOH ~-x~

3-Pyridinesulfonic acid

~

so >

(pmol rain -l mg -l)

Nicotinic acid

._> G _>

V=~x

Compounds

lO~

672

0.11

104

0.70

S O3H

4C

3-Cyanopyridine

12

150

2s

I

•

0

20

40

I

5

=

7

Temperature (°C)

I

9

11 3-Pyridylacetic acid

FIG. 2. Effects of temperature (A) and pH (B) on the stability of nicotinic acid dehydrogenase. (A) After the purified enzyme (2.0 units) was incubated at various temperatures for 10 rain, the enzyme was cooled on ice. The residual activity was measured under the standard conditions. (B) Purified enzyme (2.0 units) was incubated at 50°C for 10 rain in the following buffers (final concentration of 90 raM): sodium acetate, ©; Tris-HCl, O; potassium phosphate, i ; sodium borate, n . Enzyme solution was then cooled and pH of each enzyme solution was adjusted to 7.0 by adding 1 M potassium phosphate buffer (pH 7.0). Residual activity was measured under the standard conditions.

purified enzyme exhibited three bands (molecular masses of 85, 34, and 20 kDal) on SDS-PAGE (34). The absorption spectrum of the purified Bacillus enzyme exhibited peaks at 450 and 550 nm indicating the presence of flavin and Fe/S centers (34), and the absorption peaks at 450 and 550 nm were markedly decreased by the addition of nicotinic acid. C. barkeri nicotinic acid dehydrogenase was reported to contain molybdo-seleno-iron/sulfurflavoprotein (35-38). Thus, in these respects, P. fluorescens TN5 enzyme seems to be quite different from the B. niacini and C. barkeri soluble enzymes. Enzyme stability Although the enzyme was stable in the membrane-bound form, the solubilized enzyme was very labile. However, the soluble enzyme could be stabilized by adding nicotinic acid to make a final con-

1oo

f

gso ~ 40

C

I/

10

.= .

i

30

,

|

50

•

J

70

Temperature (°C)

6

7

8

0

i

pH

200t

.~CH2OH

3-Pyridylcarbinol

A... = 60 80

9

10

pH

FIG. 3. Effects of temperature (A) and pH (B) on the activity of nicotinic acid dehydrogenase. (A) The reactions were carried out for 10 rain in the standard reaction mixture containing 2.0 units of the purified enzyme at various temperatures. (B) The reactions were carried out for 10min at 35°C in the following buffers containing 2.0 units of the purified enzyme: 90 mM Tris-HC1, o ; 90 mM potassium phosphate, • .

-Picoline Nicotinamide

~- -C

PaCOOH C 1-13

~ N"

~f

0 0

CONHz 0

-~COOC~

Methyl nicotinate

0

~,~.~'"COOH 2-Hydroxynicotinic acid ~ ~ "

Quinolinic acid

- N - \ OH COOH

~

1

2.1

0

COOH ~ N%~COOH Pyrazinecarboxylic acid

~

N~

170

9.8

Enzyme assays were carried out spectrometricaUy in the standard reaction mixture containing 0.5 to 70 units of the purified enzyme.

centration of 20 mM. Therefore, 20raM nicotinic acid was added to all buffers used in all procedures during the course of purification. The stability of the highly purified enzyme was examined under various temperature and pH conditions. After the enzyme was preincubated for 10min in 0.1 M potassium phosphate buffer (pH 7.0) at various temperatures, the enzyme activity was assayed under standard conditions. As shown in Fig. 2A, the purified enzyme was found to be stable below 50°C. Next, the stability of the purified enzyme at various pHs was examined. After the enzyme was preincubated for 10 min in various buffers at 50°C, the remaining activity was assayed under standard conditions. The purified enzyme was found to be stable over a pH range between 5.0 and 8.8 (Fig. 2B). Effects of temperature and pH on enzyme activity The effect of temperature on the enzyme activity was examined using nicotinic acid as the substrate. The activity was measured at various temperatures over 20-70°C. The initial velocity of the nicotinic acid hydroxylation by the enzyme increased with the increase of temperature, reaching a maximum at 50°C (Fig. 3A). The effect of pH on the enzyme activity was also examined. The enzyme showed activity in a broad pH range between 4.5 and 9.0, and showed the maximum activity at around 8.3 (Fig. 3B). Substrate specificity The substrate specificity of the purified enzyme was examined (Table 2). In addition to nicotinic acid, 3-pyridinesulfonic acid and pyrazinecar-

Vot. 78, 1994 TABLE 3. Effects of various compounds on the activity of nicotinic acid dehydrogenase Compounds Relative activity (%) None 100 KCI, LiC1, NaC1, CaC12, MnC12, BaC12, MgC12, ZnC12, COC12,NiCI2, AIC13, FeC13, Na2MoO4 95-100 CuC12, HgCl2, AgNO3 0 p-Chloromercuribenzoic acid 0 N-Ethylmaleimide 0 Iodoacetate 105 5,5'-Dithio-bis(2-nitrobenzoic acid) 100 NaF 104 NaN3 83 Hydroxylamine 104 Semicarbazide 98 Phenylhydrazine 85 Cysteamine 103 a,a'-Dipyridyl 79 o-Phenanthroline 98 8-Hydroxyquinoline 100 EDTA 102 Disodium 4,5-dihydroxyl-m-benzene disulfonic acid 93 Urea 100 Acriflavin 78 After incubating the purified enzyme (2.1 units) with 1 mM of various compounds for 10 min at 35°C, the remaining activity was assayed under the standard reaction conditions. boxylic acid acted as suitable substrates, and the formation o f the corresponding hydroxyl c o m p o u n d s was detected as a new a b s o r p t i o n peak by H P L C analysis. These c o m p o u n d s were isolated and identified (Nagasawa, T. et al., A b s t r . A n n u . Meet. J a p a n Soc. Biosci. Biotech. A g r o c h e m . , J a p a n , p. 338, 1991). 3-Cyanopyridine and 2-hydroxynicotinic acid were also hydroxylated, albeit at a lower rate. On the other hand, nicotinamide, methyl nicotinate,/3-picoline, 3-pyridylcarbinol, quinolinic acid, 3-pyridylacetic acid, 6-hydroxynicotinic acid, 3hydroxypyridine and 3-chloropyridine were not hydroxylated even by adding the entire a m o u n t o f enzyme with p r o l o n g e d incubation. Thus, these findings indicate that the negative polarity at the 3-position on the pyridine ring is i m p o r t a n t for the hydroxylation reaction. lnhibitors The effects o f various c o m p o u n d s as potential inhibitors were investigated (Table 3). The enzyme activity was not influenced by the a d d i t i o n o f metal ions except A g ÷, Cu 2+ and H g 2÷. The enzyme displayed a sensitivity to thiol reagents such as AgNO3, HgC12, CuC12, p - c h l o r o m e r c u r i b e n z o a t e , and N-ethylmaleimide, suggesting that thiol groups m a y play an imp o r t a n t role for the catalytic function o f the enzyme. Metal chelating agents (e.g., o-phenanthroline, 8-hydroxyquinoline, E D T A , and d i s o d i u m 4,5-dihydroxy-m-benzenedisulfonic acid) barely influenced the activity, demonstrating that the enzyme seems to lack a metal-ion requirement. The enzyme was not sensitive to carbonyl reagents. Requirement of electron acceptors for hydroxylation of nicotinic acid W h e n P. fluorescens TN5 resting cells were incubated with nicotinic acid, the hydroxylation o f nicotinic acid proceeded efficiently without the addition o f any electron acceptors as long as complete aeration was p e r f o r m e d (27). Next, the cells were disrupted b y ultrasonication a n d the cell extract was fractionated by ultracentrifugation at 1 0 0 , 0 0 0 x g for 6 0 m i n . W h e n the supernatant solution thus o b t a i n e d was incubated with nicotinic acid, hydroxylation was barely observed

NICOTINIC ACID HYDROXYLATION

23

TABLE 4. Ability of various compounds as electron acceptor for nicotinic acid dehydrogenase Acceptor K3Fe(CN)6 Methylene blue PMS FAD FMN NAD NADP 2, 6-Dichloroindophenol Cytochrome c (horse heart) 2, 3, 5-Triphenyl-2H-tetrazolium Neutral red Safranin O Nitro blue tetrazolium Menadione

Relative activity (%) 132 0 100a 0 0 0 0 0 0 0 0 0 1.75 0

" Corresponds to 672 pmol min -1 (mg protein) -I. Various reagents (270 nmol) were added to the reaction mixture (2.7 ml) in a cuvette containing 200 pmol of potassium phosphate buffer (pH7.0), 30pmol of nicotinic acid and 0.35 units of the purified enzyme. The incubation was carried out at 35°C. The increase in the absorbance at 290 nm corresponding to the formation of 6-hydroxynicotinic acid was measured. (Fig. 4). However, in the presence o f P M S , the hydroxylation reaction o f nicotinic acid proceeded efficiently. In addition, the highly purified enzyme did not exhibit any activity without the addition o f PMS. Thus, the enzyme which catalyzed the f o r m a t i o n o f 6-hydroxynicotinic acid seemed to be a PMS-requiring dehydrogenase. On the other hand, the precipitate obtained by ultracentrifugation at 1 0 0 , 0 0 0 x g for 6 0 m i n exhibited high activity even without the addition o f P M S (Fig. 4). However, a significant enhancement o f the activity was also f o u n d with the precipitate supplemented by PMS. The highly purified enzyme p r o b a b l y lost its own native electron acceptors during the course o f solubilization and purification. On the other hand, the precipitate seems to contain its own native electron acceptors. However, the electron acceptors or the electron respiratory chains in the cells might be partially d a m a g e d during precipitation. Other-

20

•S

16

o 12

g. 2

04 08 S~ )ernatant solution (rag as protein)

80 Pre©lpltate (J~g as protein)

0

2.0 4.0 Purified enzyme (lag as protein)

FIG. 4. Effect of PMS on the formation of 6-hydroxynicotinic acid by the purified enzyme, the cell-extract supernatant solution and the cell extract precipitate. Various amounts of the purified enzyme, cell-extract supernatant solution and cell extract precipitate were incubated for 10 rain in the standard reaction mixture in the presence (closed marks) or absence (open marks) of 50 mM PMS.

24

J. FERMXNT.BIOENCI.,

HURH ET AL. TABLE 5.

Effect of PMS and KCN on the hydroxylation reaction

Reaction mixture

I

I

400

420

I

I

I

I

I

440 520 560 Wavelength (nm)

I

600

6-Hydroxynicotinic acid formed (pm01 min-r ml-r) KCN (5 mM)

No addition

0.88 10.5 0.02 1.50

4.1 10.5 0.36 1.54

Resting cells Resting cells + PMS (50 mM) Respiratory particles Respiratory particles+ PMS (50 mM)

After incubation of the resting cells (3.2 mg as dry weight) or the respiratory particles (16 mg as protein) with or without 5 mM KCN for 10 min at 35”C, the hydroxylation reaction was assayed in the presence or absence of 50 mM PMS under the standard conditions.

r

safranin 0 and menadione were inert electron acceptors. K3Fe(CN), was the most suitable electron acceptor and nitro blue tetrazolium also acted as an electron acceptor.

I J !Yi ,oo 420

I

I

440 520 Wavelength (nm)

I

I

660

600

Respiratory chain linked to nicotinic acid hydroxylation reaction by P. fluorescens TN5 The cell extract from nicotinic acid-induced P. fruorescens TN5 cells

FIG. 5. Absorption spectra of the cell extracts prepared from nicotinic acid-induced or non-induced cells. After P.fluorescens TN5 was cultivated in the medium supplemented with (B) or without (A) 81.2 mM nicotinic acid, the cell extract was prepared. Units used for measurement: A, 5.4 mg protein per ml (12.4 units/ml); B, 5.7 mg protein per ml (0.01 units/ml). wise, PMS may accelerate the electron transfer from the electron respiratory chain to oxygen. Using the highly purified enzyme, we examined the various electron acceptors for the Pseudomonas nicotinic acid dehydrogenase. As shown in Table 4, methylene blue, FAD, FMN, NAD, NADP, horse heart cytochrome c, 2,3,5-triphenyl-2H-tetrazolium, neutral red,

0.08

0.04 0" ii -E 51 2 0.00

-0.04

I

I

500

600

I

Wavelength (nm) FIG. 6. Changes in spectra by the addition of nicotinic acid and Na2S203 to the respiratory particles. (-): respiratory particles (0.36 units/ml); (.....,) and (----): 1 min and 60 min after the addition of 10 mM nicotinic acid to the respiratory particles, respectively; (----): a small quantity of Na&O, crystal was added to the respiratory particles and incubated for 10 min.

showed a reddish color, which was not observed with the cell extract prepared from the cells grown on the medium without nicotinic acid. As shown in Fig. 5, the reddish color seems to give an absorption spectrum characteristic of cytochrome c (absorption maxima at 418, 521 and 550nm). It might be plausible to conclude that nicotinic acid induces the formation of the cytochrome-linked respiratory chain as well as of nicotinic acid dehydrogenase (27, 28). In addition, cell extracts prepared from nicotinic acid-induced cells or non-induced cells were subjected to SDS-PAGE and hemeassociated peroxidase activity staining to detect the occurrence of heme-related compounds (41). Blue-stained bands were clearly observed only with the cell extract of nicotinic acid-induced cells (data not shown). These results therefore suggest that the formation of hemebound protein bands is induced by the addition of nicotinic acid to the medium. In order to elucidate the relationship between nicotinic acid dehydrogenase and the cytochrome respiratory chain, we prepared respiratory particles from nicotinic acid-induced cells as described under Materials and Methods. The absorption spectrum of the respiratory particles was measured in the presence or absence of nicotinic acid (Fig. 6). A marked Soret band at 428 nm, and additional peaks at 520 and 550nm, clearly appeared with the addition of nicotinic acid to the respiratory particles. When the respiratory particles were reduced with NazSz03, almost the same absorption spectrum appeared. The absorption spectra seem to be characteristic of cytochrome c. The linkage of nicotinic acid dehydrogenase to the cytochrome respiratory chain in P. ovalis Chester was also reported (30, 43). Next, we examined the effect of PMS and KCN on the hydroxylation reaction (Table 5). Nicotinic acid hydroxylation activity was strongly suppressed by the addition of KCN and partially restored by the addition of PMS. KCN is known to be a specific inactivator of the cytochrome respiratory chain. However, PMS can act as an artificial electron acceptor and overcome to some extent the inactivation of the cytochrome respiratory chain. We earlier confirmed that the oxygen of HZ0 is incorporated into the hydroxyl group of 6-hydroxynicotinic acid by nicotinic acid dehydrogenase of P. jkorescens TN5 through experiments using H201* and gas-mass anal-

VoL. 78, 1994

~

COOH

NICOTINIC ACID HYDROXYLATION

Nicotinic acid dehydrogenase

H~,O

~1/COOH

2[H] I I

Cytochrome respiratory chain , ~ f f ' ~ 1/2 02 I

+ H20 FIG. 7. Proposed scheme of nicotinic acid hydroxylation reaction catalyzed by nicotinic acid dehydrogenase in P. fluorescens TN5.

ysis (Omura, K. et al., Abstr. Annu. Meet. Japan Soc. Biosci. Biotech. Agrochem., Japan, p. 156, 1992). Based on these results, it can be concluded that nicotinic acid dehydrogenase is linked to the cytochrome respiratory chain and that nicotinic acid induces the formation of not only nicotinic acid dehydrogenase but also the cytochrome respiratory chain, both of which are indispensable for the hydroxylation of nicotinic acid in the cells. Thus, the proposed scheme of nicotinic acid hydroxylation reaction catalyzed by nicotinic acid dehydrogenase in P. fluorescens TN5 is outlined as shown in Fig. 7. REFERENCES 1. Theriault, R.J. and Longfield, T. H.: Microbial conversion of acetanilide to 2"-hydroxyacetanilide and 4'-hydroxyacetanilide. Appl. Microbiol., 15, 1431-1436 (1967). 2. Schwartz, R.D., Williams, A.L., and Hutchinson, D.B.: Microbial production of 4,4'-dihydroxybiphenyl: biphenyl hydroxylation by fungi. Appl. Environ. Microbiol., 39, 702708 (1980). 3. Golbeck, J. H. and Cox, J. C.: The hydroxylation of biphenyl by Aspergillus toxicarius: conditions for a bench scale fermentation process. Biotechnol. Bioeng., 26, 434-441 (1984). 4. Cox, J.C. and Goibeck, J.H.: Hydroxylation of biphenyl by Aspergillus parasiticus: approaches to yield improvement in fermentor cultures. Biotechnol. Bioeng., 27, 1395-1402 (1985). 5. Steve, S. and Brown, S.: Biotechnology yields new fine chemicals. Performance Chem., 11, 18-23 (1986). 6. Sakai, T., Shibata, M., Yabusaki, Y., and Ohkawa, H.: Expression in Saccharomyces cerevisiae of chimetic cytochrome P450 cDNAs constructed from cDNAs for rat cytochrome P450c and P450d. DNA, 6, 31-39 (1987). 7. Abramowicz, D.A., Keese, C.R., and Lockwood, S.H.: Regiospecific hydroxylation of biphenyl and analogs by Aspergillus parasiticus, p. 63-92. In Abramowicz, D.A. (ed.), Biocatalysis. Van Nostrand Reinhold, New York (1990). 8. Yoshida, S., Yoshikawa, A., and Tero, I.: Microbial production of hydroquinone. J. Biotechnol., 14, 195-202 (1990). 9. Conrad, H.E., Dubus, R., Namtvedt, M.J., and Gunsalus, I.C.: Mixed function oxidation II. Separation and properties of the enzymes catalyzing camphor lactonization. J. Biol. Chem., 240, 495-503 (1965). 10. Nagasawa, T., Yoshioka, H., and Yamada, H.: Microbial conversion of o-acetyltoluidide into 4'-hydroxy-o-acetyltoluidide. Appl. Microbiol. Biotechnol., 34, 325-329 (1990). 11. Yoshioka, H., Nagasawa, T., Hasegawa, R., and Yamada, H.: Conversion of N-acetyl-o-toluidine into 4"-hydroxy-N-acetyl-otoluidine by Fusarium verticillioides. Biotechnol. Lett., 12, 679-684 (1990). 12. Krezer, A. and Andreesen, J . R . : A new pathway for isonicotinate degradation by Mycobacterium sp. INA1. J. Gen. Microbiol., 137, 1073-1080 (1991).

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