Vol. 168, No.. 2, 1990 April 30, 1990

BIOCHEMICAL

PURIFICATION OF INACTIVATED

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 437-442

PHOTORESPON!tiIVE NITRILE HYDRATASE

Teruyuki Nagamune, Hideyuki Kurata*, Makoto Hirata*, Jun Honda*, Hiroyuki Koike**, Masahiko Ikeuchi**, Yorinao Inoue**, Akira Hirata* and Isao Endo Frontier *School

Program,

of Science and Engineering, **Solar

Received

Research

February

Energy

Research

Group,

RIKEN.

Wako-Shi,

Saitama

Waseda

Vtdversity,

Shinjuku-ku,

RIKEN,

Wako-shi,

Saitama

351-01,

Japan

Tokyo 3X-01,

169, Japan Japan

2, 1990

Summary: Photoresponsive nitrile hydratase from Rho&xoccus sp. N-771 was purified in its inactivated form. The enzyme had a molecular weight of approximately 60 kDa and consisted of 2 subunits each having molecular weight of 27.5 and 28 kDa. The enzyme also contained 2 iron atoms/enzyme as a cofactor. The enzyme was more stable in its inactivated form, rather than the activated during storage in the dark. The enzyme was most stable in the temperature region of 0-35°C and lost its activity above 40°C. The enzyme was most stable in the pH region of 6-8. The optimum temperature and pH for the enzyme activity was 30°C and 7.8, respectively. The enzyme showed wide substrate specificity, and most of the metal ions did not affect enzyme activity significantly. The absorption spectrum revealed the presence of some cofactor which changed 0 1990 Academic Press, Inc. form after photoirradiation. Aliphatic nitrile hydratase catalyzes the hydration of aliphatic nitrile to amide. Recently, it has been revealed that nitrile hydratase from Rhodococcus that when cells of Corynebacterium

shows an unusual property towards light. Nakajima et al. [l] observed

sp. N-774 (later re-identified as Rhoa’ococcus

sp N-774)

were

stored at 0°C

in the dark, nitrile hydratase activity of cells gradually decreased, and most of the lost activity of cells or extracts could he restored by irradiation with light, whereas the enzyme activity of Pseudomonas affected by light. We reported [2] that inactivation of nitrile hydratase activity of Rhodococcl*r

sp.

was

not

N-771 cells

in the dark depended on both dissolved oxygen concentration and temperature, and that the enzyme in the cells was also reactivated by light. In addition, the enzyme activity of cell-free extract of inactivated cells could also be recovered by photoirradiation, dark and aerobic incubation. Rhodococcus

though the photoactivated enzyme of the extract could not be inactivated by Endo et al. [3] purified and crystallized photoactivated nitrile hydratase of

sp. N-774 by adding n-butyrate as a compound stabilizing the enzyme, and clarified that the

activity of purified enzyme did not decrease during incubation in the dark. Our aim in this paper is to purify the photoresponsive nihile

MATERIALS

hydratase in its inactivated form, and clarify some characteristics of this unique

AND METHODS

Organism and cultivation. Rhodococcus sp. N-771 was cultivated aerobically with YM broth as previously described [21. Enzyme purification. All procedures were carried out at 5 “C in the dark. 20 mM phosphate buffer @H 7.8) was used throughout the purification except where specified. 0006-291x/90 437

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Vol. 168, No. 2. 1990

BIOCHEMICAL

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RESEARCH COMMUNICATIONS

Step 1. Preparation of cell-free extract. Cell suspension (100 g wet cell in 500 ml of buffer) was incubated aerobically in the dark at 5 “C for 24 hours. During this time the activity of nitrile hydratase decreased by about 95-98 %. ‘Ihe inactivated cells were treated with 1 g lysozyme (Sigma, grade I) at 20 “C for about 10 hours. The cell suspension was removed by centrifugation at 27,170 x g for 30 minutes. The supematant was used as the crude cell-free extract. Step 2. Ammonium sulfate fractionation. The crude cell-free extract was brought to 40 % saturation by gradually adding crystalline ammonium sulfate and continuously stirring. After standing for 30 minutes, the resulting precipitate was removed by centrifugation at 27,170 x g for 30 minutes. Ammonium sulfate was further added to the supematant up to 60 % saturation. After 30 minutes, the precipitate formed was collected by centrifugation at 27,170 x g for 30 minutes, dissolved in buffer and then dialyzed in buffer overnight. Step 3. DEAE-cellulose column chromatography. The dialyzed enzyme solution was loaded to a DEAEcellulose column (2.5~100 cm, Whatman DE-52) which had been equilibrated with buffer. The enzyme was eluted with a linear gradient of Tris-HCl buffer @H 7.8, 0.3 - 0.7 M) at a flow rate of 120 ml/b. Step 4. Hydrophobic column chromatography. The enzyme solution from step 3 was loaded to a hydrophobic column (2.5~100 cm, Toyopearl HW-65C) which had been equilibrated with buffer containing 40 % saturated ammonium sulfate. The enzyme was eluted by lowering the ionic strength of ammonium sulfate (1.3 - 0.5M) in buffer at a flow rate of 150 ml/h. The active fractions were collected, dialyzed against buffer and concentrated by ultrafiltration with Diaflo ultra8ltration membrane PM10 (Amicon). Step 5. DEAE-cellulose column chromatography (2nd). The enzyme solution was loaded to a DEAEcellulose column (1.4~20 cm, Whatman DE-52) which had been equilibrated with buffer. The enzyme was eluted with a linear gradient of Tris-HCl buffer (pH 7.8, 0.3 - 0.7 M) at a flow rate of 12 ml/h. Active fractions were collected. Step 6. Gel filtration. The enzyme solution was loaded to a gel filtration column (1.5X100 cm Sepharose CL6B) equilibrated with buffer and eluted with buffer at 6 ml/h. The active fractions were collected and concentrated as in step 4. Electrophoresis. SDS-gel electrophoresis was performed by a method of Laemmli [4]. Molecular weight. The molecular weight of the purified niuile hydratase was estimated by HPLC. The enzyme was subjected to HPLC on a TSK-3000 SW protein column 7.5 x 600 mm, (Tosoh). The elution of the enzyme was carried out with 0.4 M phosphate buffer (pH 7.5) with a flow rate of 0.5 ml/min at 25°C. The absorbance of the effluent was monitored at 280 nm. The molecular weight of the enzyme was calculated from the elution volume of the standard proteins: pyruvate kinase (237 kDa), lactate dehydrogenase (132 kDa), glucosedphosphate dehydrogenase (128 kDa), malate dehydrogenase (67 kDa), bovine serum albumin (67 kDa), ovalbumin (45 kDa), a-chymotrypsinogen A (24 kDa) and cytochrome C (13 kDa). Metal Analysis. Prior to analysis, the purified inactive and photoactivated enzymes were dialysed against 2 changes of 20 mM phosphate buffer for 24 hours. The samples containing 0.5 - 1.0 mg enzyme/ml was measured with an inductively coupled radiofrequency plasma spectrophotometer, Shimadzu ICPS-50. The metal contents of the enzyme were determined from the calibration curves of standard solutions. Photoirradiation and enzyme assay (standard condition). An appropriate amount of the enzyme in 0.50 ml of 20 mM phosphate buffer @H 7.8) in a glass vial was exposed to 5000 lx light (71.0 W/m*) from 500W Toshiba photoreflector lamp “SPOT” for 10 minutes in an ice bath. Then 0.45 ml of 1 M propionitrile was added, kept in an ice bath for 1 minute and 0.05 ml 2N HCI was added to stop enzyme reaction. Amount of propionamide formed was assayed by gas chromatography under the condition described previously [2]. Protein was determined by the modiied Lowry’s method developed by Besadoun et al. 151 with bovine serum albumin (Seikagaku Kogyo) as a standard. Protein was determined from the absorption at 280 nm and the absorption coefficient was calculated to be 1.51 mg‘t ml cm-l by absorbance and dry-weight determinations. A unit of enzyme is defined as the amount of enzyme which forms lpmol of propionamide per minute and the specific activity is expressed as units of enzyme per mg protein. Enzyme stability. The purified inactive enzyme could be reactivated by photoirradiation. Both inactive and photoactivated enzymes were incubated in 50 mM phosphate buffer @H 7.8) at 5 OC in the dark aerobically to examine the stabilities of the enzymes. The inactive puritied enzyme was sampled and photoirradiied just before the enzyme assay to estimate its activity. The photoactivated enzyme which was initially irradiated and incubated in the dark was used to examine the activity loss in the dark incubation and the recovery of it by reirradiation. The stability of the photoactivated enzyme was examined under various pH conditions. Piit, optimum pH was determined by assaying the enzyme activity at various pH of the following buffers (final concentration of 50 ml@ sodium acetate buffer (pH 5 - 6). phosphate buffer (pH 6.2 - lo), Tris-HCl buffer (PH 8.5 - 10) and glycine buffer @H 10 - 10.5). Then after the enzyme was incubated at 0 “C for 10 minutes in the buffers just mentioned, enzyme activity was assayed at the determined optimum PH. The stability of the enzyme was examined at various temperatures. The inactive and photoactivated enzymes were incubated at various temperatures for 10 minutes and assayed under standard condition. The inactive enzyme was phou&mdiated for 10 minutes just before the assay. Substrate specificity. The enzyme activity was assayed with various substrates under standard condition in order to examine substrate specificity. 438

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Table 1. Summaryof purification of inactive nitrile hydratasefrom Rhodococcus Total activity

l.Cell-free exnact 2.Ammonium fractionation

sulfate

3.DEAEcellulose 4.Toyopearl t-W-6X S.DEAE-cellulose (2nd) 6.Seohamse CL-6B

protein

Specific activity

Yield

[uhgl

WI

sp. N-771

Purification fold L-1

WI 399,170

[mgl 9,970

40

100

1.0

226,610

2,215

102

57

2.6

188,840 116,780 96,369 79,480

780 208 150 115

242 561 642 691

47 29 24 20

6.1 14.0 16.1 17.3

Effect of metal ions. Effect of metal ions on enzyme activity was observed with various reagents containing each metal. They were added to each assay mixtures so that the final concentration of metal ion was 0.5 mM. Absorption and difference spectra. Absorption and difference spectra of inactivated and photoactivated enzymes were measured in crystal cuvettes of 1 cm path length with Shimadzu MPS-2000 spcctrophotometer at room temperature. RESULTS AND DISCUSSION Enzyme purification

and analysis. The overall enzyme purification achieved was about 17-fold with an yield

of 20 %. The result of the purification is summarized in Table 1. Elcctrophoresis of the purified enzyme showed 2 adjacent bands (Pig. 1) and the molecular weight of each being 27.5 kDa (a subunit) and 28 kDa (p subunit). Photoirradiated

purified enzyme gave the same results. From the results of HPLC, the purified

holoenzyme was estimated to have a molecular weight of about 60 kDa. Metal analysis, Qualitative analysis of metals showed that inactivated and photoactivated enzyme contained iron. Plasma emission spectroscopy of the enzyme solutions revealed that the enzymes contained 2 atoms of

AB

C

D

E

F

1

2

01 +

-

3

02

0

200

400

Time

600

do

[h ]

SDS-polyacrylamide gel electrophoresis of purified inactive nitrite hydra&se. Lane 1: 5 pg purified nitrile (14.4 kDa) B-soy beau hydratase.photoirradiated just before loading. Lane 2: marker proteins A-a-lactalbumin

F&l-.

trypsin inhibitor

(20.1 kDa) C-carbonic

anhydrase

(30 kDa) D-ovalbumin

(43 kDa) E-bovine

serum albumin (67

kDa) F-phosphorylase.b (94 IcJJa)Lane 3: 5 pg purified inactive nilrile hydratase. w Stability of inactive nitrile hydratase ( 0) and photoactivated nitrile hydratase ( 0). These enzymes were stored in 5OmM phosphate buffer @H 7.8) in the dark at 5°C. The inactive enzyme was irradiited for 10 minures just before the assay under the standard condition.

439

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BIOCHEMICAL

Temperature A

40

30

3.2

3.3

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

[“Cl

20

10

0

_

15 -

3A

3.5

. 3.6

&

x 10-3

l/T I K-’ 1

PB

Fig.3. The optimum temperature (A) and pH (B) for the activity of photoactivated nitrile hydratase. For sum temperature measurement, enzyme reaction rate (propionamide formed per minute in the standard assay solution) was measured for various temperatures and plotted logarithmically against the inverse of absolute temperature (Arrhenius plot). For optimum pH measurement,the enzyme was assayedin sodium acetatebuffer (0). phosphate buffer (0). Tris-HCI buffer (A) and glycine buffer (0).

Stability of enzyme. As shown in Fig. 2, the inactive purified enzyme was stable and kept 80 % of tbe initial activity after 800 hours storage at 5 “C, whereas the photoactivated purified enzyme was much more labile and lost its activity completely after 800 hours storage. In addition, the recovery of its activity by reirradiation was not observed. Effect of temperature

and pH. The optimum temperature of the enzyme activity was found to be 30 “C (Fig.

3A). The enzyme exhibited optimum activity at pH 7.8 (Fig 3B). When the photoactivated enzyme was incubated in 50 mM phosphate buffer @H 7.8) at various temperatures for 10 minutes, the enzyme activity was most stable in the range of 0 - 35 “C, but it rapidly lost its activity above 40 “C (Fig. 4A). The figure also shows the activity of inactive enzyme, which was incubated at various temperatures for 10 minutes and then irradiated for 10 minutes, was more stable than that of photoactivated one. The photoactivated enzyme under various pH was found to be most stable at pH 6 to 8, but it was labile below pH 5.5 or above pH 8.5 (Fig. 4B). From these results, it was revealed that the stability of inactive enzyme was higher than that of photoactivated one, and was kept for more than 800 hours without an addition of stabilizing reagent like n-butyric acid. This

B

0

Temperature

[ ‘C ]

'

5

'

0

m

7

'

8

'

9

'

10

'

11

PH

Fig.4.Effect.s of temperature (A) and pH (B) on the stability of photoactivated nitrite hydratam. The inactive(O) andphotoirradiated(0) enzyme samples were incubated at various temperatmes for 10 mhmtes. The inactive enzyme was irradiated for 10 minutes just before the assay under the standard condition. For the effect of pH, the enzyme was assayed in sodium acetate buffer (0). phosphate buffer ( 0 ), Tris-HCl buffer ( A) and glycine buffer ( 0 ). 440

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Table 2, The enzyme specificity on various substrates.The substrate-swere added and assayed under standardcondition with approximately 6.7 pg of enzyme Relative Substrate Activity[%] Acetoniuile 4 Propioniuile 100 n-Butyronitile 113 Isobutyronitrile 31 n-Valeronitrile 0 Acrylonitrile 170 Methacrylonitrile 75 Chloroacetonitrile 492 -

suggests that a structural change in the active site of the enzyme may have occurred after photoirradiation giving rise to unstability of the enzyme. Substrate specificity. The enzyme showed wide specificity (Table 2). Especially, it was more reactive with unsaturated substrates such as acrylonitrile and methacrylonitrile.

The substrate chloroacetonitrile

showed the

highest activity tested. This result is quite different from that of nitrile hydratases of other organisms [6,7,8] suggesting difference in the active site structures of each. Effect of metal ions. As shown in Table 3, most of the metal ions had no considerable effect on the enzyme activity except silver, which completely inactivated the enzyme. The cadmium and zinc ions both inactivated the enzyme by about 50%. Silver ion also inactivates nitrile hydratases of other organisms [6,83, but cadmium and zinc ions have no considerable effect on their enzyme activity. This result is also an indication of the difference in the active site smctures of these enzymes. Absorption

spectrum.

T’he visible and ultraviolet (UV) absorption spectra of inactive and photoactivated nitrile

hydratase at pH 7.8 are shown in Fig. 5A. The difference absorption spectrum of photoactivated enzyme against inactive one is shown in Fig. 5B. The UV spectrum of the inactive nitrile hydratase shows a typical absorption

Table 3, Effects of various metal ions on the enzymeactivity. The ions were added to the assaymixtures so that their final concentration in the reaction mixtures was 0.5 mM, except silver where it was 0.005 mM. Approximately 6.7 kg enzyme was in each mixture and incubation time was 10 minutes Reagent None AlCl,.qO BaCI$I-$O C4lCl,.‘&O CaC’2.2~0 CoC’2.2~0 CuC’2.2~0 FeCL,.6%0 FeS04.71Q0 PbCl,

Relative Activity[%] 100 120 93 49 118 114 85 97 109 108

Reagent LiCl MgC$.qO MnC$.4~0 NiC‘2 KC1 &NO3 NaCl SnC$.2I+O ZnC$

441

Relative Activity[%] 102 109 93 102 101 0 102 109 51

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2.0

1.0

8 9

O2

D

300

400

600

I

O0

Wavelength [ nm ] w Absorption spectmm (A) for inactive (-) and photoirradiated (---) nitrile hydmtasetaken over the range of 240 - 800 nm. The enzyme concentration was approximately 1.2 mg/ml. Difference spectmm (Et) of photoirradiated - inactive nitrile hydratare taken over the range of 300 - 800 nm.

maximum at 280 nm, a minimum at 250 nm and characteristic shoulder at 225 nm (not shown). In the visible range, the inactive enzyme exhibits absorption in a broad region (310 - 800 nm) with absorption maximum at 370 nm. The absorption spectrum of the photoactivated enzyme in the visible range differs completely from that of the inactive enzyme, and shows its peak at 680 nm and its shoulders at 370 nm and 450 nm. The absorption spectrum of the photoactivated enzyme was very similar in the shape and position of the peak to those of

Brevibucterium [7] and Pseudomonus [81. It can be observed in Fig. 5B that the difference absorption spectrum has a positive peak at 680 nm and nagative peaks at 280 nm (not shown) and 370 nm. The changes in absorbance at 370 mu and 680 nm showed good correlation with the increase of enzyme activity during phoroirradiation

(not shown). The occurrence of dramatic absorbance change during photoactivation - the

appearance of new absorption band at 680 MI in accordance with the diminution of the band at 370 nm suggests a conversion by photoirmdiation

of some cofactor from one form to another. These absorption peaks

of 370 nm (inactive enzyme) and 680 nm (photoactivated enzyme) probably originate from the absorption of non-heme iron center. No substantial absorption bands corresponding to heme or flavin were observed in the visible absorption spectra of inactive and photoactivated nitrile hydratase. ACKNOWLEDGMENT We thank Nitto Chemical Industry Co. Ltd. for generously providing Rhodococcus sp. N-771 and financial support. REFERENCES 1. Nakajima, Y., Doi, T., Sato, Y., Fujiwara, A. and Watanabe, I. (1987) Chem. Le#. 9, 1707-1770 2. Nagamune.$T.. Kurata, H., Hiram, M., Honda, J.. Hirata, A. and Endo, I. (1990) Phorockm. Photobid. 51, 87-90 3. Endo, T and Watanabe, 1.(1989) FEBS L&f. 243,61-64 4. Laemmli, U.K.(1970) Nature 227,680-685 5. Besadoun, A. and Weinstein D.(1976) Anal. Biuchem. 70,241-250 6. Asano, Y.. Fujishiro, K., Tani, Y. and Yamada, H. (1982) Agric. Biol. Chem. 139, 1305-1312 7. Nagasawa, T., Ryuno, K. and Yamada, H. (1986) Biochem. Biophys. Res. Commwr. 139, 1305-1312 8. Nagasawa, T., Nanba, H., Ryuno, K., Takeuchi, K. and Yamada, H. (1987) Eur. J. Biochem. 162,691-698

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Purification of inactivated photoresponsive nitrile hydratase.

Photoresponsive nitrile hydratase from Rhodococcus sp. N-771 was purified in its inactivated form. The enzyme had a molecular weight of approximately ...
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