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FEMS Microbiology Letters 59 (1989) 241-246 241 Published by Elsevier FEM 03572 Spectrophotometric identification of 8-hydroxy-5-deazaflavin" NADPH oxidoreductase activity in streptomycetes producing tetracyclines Jana Novotnfi, Ji[i Neu~il and Zden~k Ho~ifilek Institute of Microbiology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia Received 3 January 1989 Accepted 31 January 1989 Key words: 8-Hydroxy-5-deazaflavin; Coenzyme F420; Fragment F0; Tetracyclines; Streptomyces aureofaciens; Streptornyces rirnosus 1. SUMMARY Cell-free extracts of vegetative mycelia of Streptomyces aureofaciens and Streptomyces rimo- sus were found to reduce streptomycete-origin 8-hydroxy-5-deazaisoalloxazine derivatives (SF420) using NADPH as a donor of hydrogen and elec- trons. 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F0) also was a substrate, although with a lower reaction rate than that for SF420. NADH could not substitute for NADPH. The Fa20-reductase activity was also observed in homogenates of S. aureofaciens spores. 2. INTRODUCTION A prokaryotic chlortetracycline-producing actinomycete Streptomyces aureofaciens was the first organism from which the cosynthetic factor 1 Correspondence to: Jana Novotn~t, Institute of Microbiology, Czechoslovak Academy of Sciences, Videfiskh 1083, 142 20 Prague 4, Czechoslovakia. (CF-1) was isolated [1] and only later identified as 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F0) [2]. F 0 is an abbreviated side chain-containing derivative of redox coenzyme F420, isolated and biochemically characterized in methanogenic [3-5] and nonmethanogenic archaebacteria [5-7]. The use of mutants blocked in various steps of bio- synthesis of the antibiotic allowed establishment of the role of CF-1 for biological reduction of 5a,lla-dehydrochlortetracycline to chlortetracyc- line [1]. Later, indirect evidence indicated the par- ticipation of CF-1 in the final reduction step of oxytetracycline biosynthesis in S. rimosus [8]. The presence of 8-hydroxy-5-deazaflavins as inten- sively yellow, blue-green fluorescent compounds in both S. aureofaciens and S. rimosus (their con- tent being minimally 20.2 and 4.5 pmol per mg dry cell weight, respectively) was reported in a study demonstrating a widespread distribution of these substances among Streptomyces species and related bacteria [9]. A different coenzymatic role of 8-hydroxy-5- deazaisoalloxazine chromophore was shown in S. griseus, where it was found to act as a photon acceptor in a DNA-photoreactivating enzyme in- 0378-1097/89/$03.50 © 1989 Federation of European Microbiological Societies

Spectrophotometric identification of 8-hydroxy-5-deazaflavin: NADPH oxidoreductase activity in streptomycetes producing tetracyclines

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FEMS Microbiology Letters 59 (1989) 241-246 241 Published by Elsevier

FEM 03572

Spectrophotometric identification of 8-hydroxy-5-deazaflavin" NADPH oxidoreductase activity in streptomycetes

producing tetracyclines

J a n a Novotnf i , Ji[i Neu~i l and Zden~k Ho~ifi lek

Institute of Microbiology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia

Received 3 January 1989 Accepted 31 January 1989

Key words: 8-Hydroxy-5-deazaflavin; Coenzyme F420; Fragment F0; Tetracyclines; Streptomyces aureofaciens; Streptornyces rirnosus

1. SUMMARY

Cell-free extracts of vegetative mycelia of Streptomyces aureofaciens and Streptomyces rimo- sus were found to reduce streptomycete-origin 8-hydroxy-5-deazaisoalloxazine derivatives (SF420) using N A D P H as a donor of hydrogen and elec- trons. 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F0) also was a substrate, although with a lower reaction rate than that for SF420. N A D H could not substitute for NADP H. The Fa20-reductase activity was also observed in homogenates of S. aureofaciens spores.

2. I N T R O D U C T I O N

A prokaryot ic chlor te t racycl ine-producing actinomycete Streptomyces aureofaciens was the first organism from which the cosynthetic factor 1

Correspondence to: Jana Novotn~t, Institute of Microbiology, Czechoslovak Academy of Sciences, Videfiskh 1083, 142 20 Prague 4, Czechoslovakia.

(CF-1) was isolated [1] and only later identified as 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F0) [2]. F 0 is an abbreviated side chain-containing derivative of redox coenzyme F420, isolated and biochemically characterized in methanogenic [3-5] and nonmethanogenic archaebacteria [5-7]. The use of mutants blocked in various steps of bio- synthesis of the antibiotic allowed establishment of the role of CF-1 for biological reduction of 5a, l la-dehydrochlortetracycline to chlortetracyc- line [1]. Later, indirect evidence indicated the par- ticipation of CF-1 in the final reduction step of oxytetracycline biosynthesis in S. rimosus [8]. The presence of 8-hydroxy-5-deazaflavins as inten- sively yellow, blue-green fluorescent compounds in both S. aureofaciens and S. rimosus (their con- tent being minimally 20.2 and 4.5 pmol per mg dry cell weight, respectively) was reported in a study demonstrating a widespread distribution of these substances among Streptomyces species and related bacteria [9].

A different coenzymatic role of 8-hydroxy-5- deazaisoalloxazine chromophore was shown in S. griseus, where it was found to act as a photon acceptor in a DNA-photoreact ivat ing enzyme in-

0378-1097/89/$03.50 © 1989 Federation of European Microbiological Societies

242

volved in the splitting of UV-induced pyrimidine dimers [10]. The family of 8-hydroxy-5-deazaisoal- loxazine derivatives purified from S. griseus (SF420) has been proposed to be a mixture of three components that possess an identical chromo- phore, but are different in the length of the lactyl- oligoghitamyl side chain [11]. Existence of a specific NADPH: 8-hydroxy-5-deazaisoalloxazine oxidoreductase isolated from S. griseus was re- ported [12].

Despite the proposed redox function of 8-hy- droxy-5-deazaflavin(s) in the tetracycline-produc- ing streptomycetes, the enzymes serving for the transfer of electrons from and to 5-deazaflavins have not been identified yet, though the activity of tetracycline dehydrogenase, the last enzyme in the biosynthetic pathway of tetracyclines, was de- tected in cell-free extracts of S. aureofaciens [13,141.

We report here the occurrence of oxidoreduc- tases in S. aureofaciens and S. rimosus, that are able to reduce the streptomycete-origin SF4z 0 with electrons generated from N A D P H and that might be the enzymes responsible for supplying the terminal biosynthetic step catalysed by tetra- cycline dehydrogenase with reducing equivalents.

3. MATERIALS A N D M E T H O D S

3.1. Organisms and cultivation Spores of Streptomyces aureofaciens 84/25 were

grown on M1 agar plates covered with cellophane and harvested after 14 days [15]. Dormant un- treated spores were stored at - 2 6 °C until disin- tegration.

Liquid cultures of this strain were grown in a complex medium with soy-bean extract and sucrose [16]. Streptomyces rimosus P was grown in the same liquid medium but sucrose was replaced by glucose. Mycelial cells were harvested after 24 h of cultivation by centrifugation, washed twice with distilled water and stored frozen.

3.2. Preparation of cell-free extracts Spores were ground with glass beads in a

mortar, extracted with 0.01 M Tris-HC1 buffer (pH 7,4) containing 10 mM magnesium acetate, 60

mM NH4C1, 6 mM 2-mercaptoethanol, 3 mM phenylmethylsulphonyl fluoride (PMSF) and centrifuged according to described methods [17].

Extracts of mycelial cells were prepared essen- tially as described elsewhere [16], except that the broken mycelia were suspended in 0.18 M Tris-HC1 buffer (pH 7.4) with 15% glycerol (v/v) , 2 mM E D T A , 0.2 m M P M S F and 2.8 m M mercaptoethanol and centrifuged at 12000 × g.

Fa20-reductase activity was retained at least for 1 year when stored at - 2 6 °C. Prior to enzyme assays all extracts were run through Sephadex G-25 columns equilibrated with 0.02 M Tris-HC1 buffer (pH 7.4).

3.3. Enzyme assay The conversion of oxidized 8-hydroxy-5-

deazaflavins to corresponding reduced forms was monitored in a reaction mixture containing 80 mM Tris-HC1 buffer (pH 7.4), 0.24 mM N A D P +, 0.6 mM glucose 6-phosphate, 1.1 U glucose 6- phosphate dehydrogenase, appropriate amount of cell-free extract and approximately 5/~M SF420 or F 0 in a total volume of 0.5 ml at 37°C. The reaction was initiated by the addition of substrate. The absorbance changes were followed in the range of 250 to 550 nm using a Hewlett-Packard 8451A diode-array spectrophotometer assisted by a pro- gram written in BASIC. The molar absorptivity of c420 = 4200 lmol -~ m m 1, taking into account the effect of p H [18], and initial slopes of reaction kinetic curves served for the calculation of F420-re- ductase activity. Concentration of the substrate was determined spectrophotometrically using the same absorptivity value.

Protein content was determined by the Coomassie blue binding assay [19] with bovine serum albumin as a standard except for S. rimosus for which the spectral method was used [20].

3.4. Chemical reduction and reoxidation of SF420 Small amounts of solid sodium dithionite were

added to a cuvette with a solution of SF420 in 0.02 M Tris-HCl buffer (pH 7.4) and absorption spec- tra were read every 10 s similarly to the enzyme assay, until the reduction was complete. Small amounts of ammonium persulphate were added

and reoxidation observed after a pH adjustment to 8 [11].

3.5. HPLC of 8-hydroxy-5-deazaflauins HPLC analyses of SF4: o and F 0 used as sub-

strates of F4:0-reductase were performed as de- scribed by Gorris et al. [21]. Runs were carried out at 28°C on a Separon ODS glass-pack column, 150 × 1 mm id, 5 #m particles (TESSEK, Prague, Czechoslovakia). The column was equilibrated with a 27.5 mM acetic ac id -NaOH buffer (pH 4,5) (phase A) and the samples were eluted, at 0.2 ml/min, with a 50-min linear gradient of 10-50% phase B (30% acetonitrile in phase A). Detection was achieved using a Hewlett-Packard diode-array detector (190-600 nm).

3.6. Materials SF4: o isolated from S. griseus and synthetic F o

were gifts from Dr. A.P.M. Eker, Delft University of Technology, Delft, The Netherlands.

4. RESULTS AND DISCUSSION

4.1. Reduction of 8-hydroxy-5-deazaflauins Spectral properties of 8-hydroxy-5-deazafla-

vins, above all the high molar absorptivity of the oxidized form ( ) k m a x = 420 nm) at alkaline and neutral pH and its conspicuous change after the reduction [11,18,22] permit monitoring of the cor- responding redox reactions spectrophotometrically [7,23-25]. Since F420 was found to be photolabile [22], the development of the method for estimating the activity of F4:0-dependent redox enzymes using a diode-array spectrophotometer had to include the evaluation of the effect of the assay light regime on the stability of the substrate during the assay. Monitoring the absorption spectra (mea- surement with 1-s integration time involving five 0.1-s measurements and 10-s repeat time) took 5 min, which was a usual time interval adopted for the enzyme-catalyzed reactions (see below). Neither the absorbance of SF4:0 nor that of F 0 dissolved in 0.5 M Tris-HC1 buffer or in a reaction mixture prepared for the enzyme assay were found to change due to the intermittent illumination of the solution in the cuvette. Consequently, instabil-

243

fq

1::1395 6 0

5 18 15 2 0 Time ( m i n )

1 5 8 "

1 8 8 "

3 9 5

582

5 I O 1 5 20 T i m e ( r n i n )

I3

Fig. 1. SF420 reduction by S. aureofaciens mycelial cell-free extract (71/~g protein). A, Absorption spectra recorded during enzyme reaction at 1-min intervals (1-6) and plotted after subtraction of the spectrum of reaction mixture without SF420; B, time course of the reaction at the absorption maxima of the substrate and product (unprocessed absorbance values are shown). 16 /~M SF420 was used, other details see MATERIALS

AND METHODS.

ity of the chromophore used is highly improbable under the conditions of the assay.

The reduction and reoxidation of SF420 were carried out essentially according to Eker et al. [11] using dithionite as the reducing agent. The reac- tions were monitored as a significant absorption decrease (Xm~ = 420 rim) and its subsequent in- crease to the original level (data not shown). The reduced form of 8-hydroxy-5-deazaflavin, how- ever, could not be detected because of a high absorption of dithionite in the interval of wave- lengths below 400 nm.

After the addition of SF420 to the cell-free ex- tract of S. aureofaciens in the presence of an NADPH-regenerating system, the rapid decrease of the absorbance at 420 nm and its parallel increase at 320 nm indicated the enzymatic reduc- tion of SF4z 0 (Fig. 1). The diode-array spectropho- tometer allowed us to measure the changing con- centrations of the substrate and product at the same time and to exclude other non-redox ab- sorbance changes.

244

.7

.5

.5

. 4 LLJ (-I

<m.2

, 1 L

i I !

^

! I i

YAVELENGTH (rm)

0

- . 1 !

1.!

.1

I@

B

f f

I I I I 1

320 nm

2 3 4 5

TI~ (mlrO Fig. 2. HPLC analysis of 8-hydroxy-5-deazaflavins. The profiles shown are for 81::420 (A) and F o (B) used as the substrates of

F420-reductase. Details, see MATERIALS AND METHODS.

4.2. Specificity of S. aureofaciens enzyme When the NADPH-regenerat ing system was re-

placed by N A D P H only, the reduction of 8-hy- droxy-5-deazaflavins was also observed. So, the function of glucose 6-phosphate as a direct elec- tron donor can be excluded. No absorption de- crease at 420 nm was found when N A D P H was replaced by NADH. The enzyme is thus similar in its N A D P H specificity to the archaebacterial [7,25,26] and S. griseus [12] oxidoreductases.

HPLC analysis of SF420 showed three main peaks with identical absorption spectra (Fig. 2) which is in agreement with the thin-layer electro- phoretic analysis of Eker et al. [11]. Since a com- plete disappearance of absorbance (~max420 rim) was observed (Fig. 1), it is suggested that S. aureofaciens F42o-reductase is able to reduce all three main components of SF42 o differing in the number of glutamyl residues. Cell-free extracts could also reduce F 0, but the activity appeared to be at least five times lower than that for SF420 (Table 1). Transient high increases of the ab- sorbance at 420 nm that are difficult to explain were observed with F o as the substrate in some experiments (data not shown). The true substrate

specificity of the enzyme will have to be de- termined by comparing the kinetic constants ob- tained with a purified enzyme. The other oxidoreductases described in the literature do not exhibit a strict specificity with respect to 8-hy- droxy-5-deazaisoalloxazine side-chain [12,26].

4.3. Enzyme distribution S. aureofaciens producing chlortetracycline and

S. rimosus producing oxytetracycline have been found to contain an enzyme which is able to reduce SF420 in vitro (Table 1) as found also for

Table 1

F420-reductase activity in streptomycetes producing tetra- cyclines estimated by using streptomycete-origin 8-hydroxy-5- deazaisoalloxazine derivatives (SF420) and 7,8-didemethyl-8- hydroxy 5-deazariboflavin (F0) as substrates

Microorganism Specific activity (nkat mg 1 protein)

Vegetative mycelium Spores

SF420 F0 SG2o F0 S. aureofaciens 0.77 0.11 1.96 0.44 S. rimosus 0.43 ND ND ND

ND, not determined.

some other microorganisms conta in ing 8-hydroxy- 5-deazaflavins [7,12,27].

The activity of F420-reductase was observed not only in a crude extract of vegetative S. aureofaciens mycelium bu t also in homogenates prepared from dorman t spores (Table 1), where the specific activ- ity of the enzyme was higher. The different specific activities could reflect various procedures for pre- par ing extracts (see MATERIALS AND METHODS) and also different aggregation of spore and vegetative ribosomes upon centr i fugat ion [17], which would appear mainly in the content of total prote ins in respective preparations. However, the presence of F420-reductase in dorman t spores suggests some impor tan t role(s) of the enzyme in the life cycle of the bacterium, since one could expect that dis- pensable enzymes would not be stored dur ing sporogenesis and dormancy.

Unt i l now, only two F420-dependent electron transferring enzymes have been demonst ra ted in microorganisms outside methanogens: oxidore- ductases of S. griseus [12] and Halobacterium cutirubrum [27]. Tet racycl ine-producing strep- tomycetes are thus new examples of aerobic organisms which are equipped with these enzymes and which feature, in addit ion, a documented metabolic step coupled to F420-dependent electron transfer. The purif icat ion of the S. aureofaciens enzyme, current ly under way in our laboratory, will enable us not only to characterize it in detail, but, in particular, to test its role in the terminal step of tetracycline biosynthesis.

A C K N O W L E D G E M E N T S

The authors wish to thank Dr. A.P.M. Eker of Delft Univers i ty of Technology for k indly supply-

ing samples of SF420 and F 0 and Dr. J. Weiser from the Ins t i tu te of Microbiology, Prague for preparat ion of S. aureofaciens spore homogenates.

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245

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