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Investigation of acetyl-CoA: Deacetylcephalosporin C O-acetyltransferase of Cephalosporium acremonium
Journal
of Biotechnology,
3 (1985)
109
109-117
Elsevier JBT 00171
Investigatioin.of acetyl-CoA: Deacetylcephalosporin C O-acetyltransferase of Cephalosporium acremonium Alfred
Scheidegger ‘** Alois Gutzwiller r, Martin Armin Fikhter 2 and Jakob Niiesch’
’ Research Laboratories .’ Institute for
of the Pharmaceutical Division of Ciba - Geigy Ltd., Biotechnology, ETH - Hiinggerberg, CH - 8093 Zurich,
T. Kienzi
‘,
CH - 4002 Baste, Switzerland
and
(Received 3 April 1985; accepted 22 June 1985)
A new, rapid test system to measure the activity of acetyl-CoA : deacetylcephalosporin C 0-acetyltransferase (DAC-acetyltransferase) was established. The ieaction product cephalosporin C could be easily analyzed by HPLC. The DAC-acetyltransferase was partially purified by means of fractionated (NH&SO, precipitation, Sephadex G-100 gel chromatography and isoelectric focusing. Molecular weight was determined to be 70 000 f 5 000 and the ~14.3 f 0.2. Besides the two substrates acetyl-CoA and deacetylcephalosporin C, no other factor necessary for the reaction could be found. The enzyme was inhibited by coenzyme A (61%), deacetoxycephalosporin C (57%), penicillin N (14%), 2,6dihydroxybenzoic acid (27%) and pyruvate (37%) at a concentration of 0.1 mM in each case. Cephalosporium
acremonium,
deacetylcephalosporin
C, acetyltransferase,
enzyme
characterization
Introduction
One major problem
of the bioproduction of cephalosporin C (CephC) with is the accumulation of considerable amounts of the precursor deacetylcephalosporin C (DAC) during the later part of the cultivation Cephalosporium acremonium
* Present
address:
0168-1656/85/$03.30
Institute for Chemical Research, Kyoto University, Uji, Kyoto-fu 611, Japan. 0 1985 Elsevier Science Publishers B.V. (Biomedical Division)
110
(Huber et al., 1968). The excreted DAC leads to a loss in the CephC yield. DAC is formed either via the biosynthetic route from deacetoxycephalosporin C (DAOC) or by enzymatic and chemical hydrolyzation of CephC. Under well-controlled cultivation conditions high yielding strains will only release low concentrations of esterase activity. Thus, the large amounts of DAC found are most probably the result of an incomplete biosynthesis of CephC. To be able to control positively the acetylation reaction, knowledge of the enzyme involved is a prerequisite. The presence of the catalysing enzyme acetyl-CoA : DAC o-acetyltransferase (DAC-acetyltransferase) was demonstrated in cell free extracts as well as in permeabilized cells by measuring the rate of incorporation of [1-‘4C]acetyl-CoA into CephC (Fujisawa and Kanzaki, 1975; Liersch et al., 1976; Felix et al., 1980). Either acetyl-CoA or acetate plus coenzyme A and ATP served as the acetyl donor. Fujisawa and Kanzaki (1975) described the enzyme to be dependent on Mg2+ as a cofactor, whereas Liersch et al. (1976) could not find such a dependency. The enzyme test method used by these authors has the disadvantage of being time consuming and limited in accuracy and determinations of kinetic data and a characterization of the enzyme are hardly possible. To investigate the properties of the enzyme and in order to obtain a more detailed understanding of the regulation of the terminal reaction in the biosynthetic pathway of CephC, a more precise and rapid enzyme test system is required. This paper deals with a new system for measuring DAC-acetyltransferase activity, the partial purification of this enzyme and its characterization and regulation.
Materials and Methods Materials
Acetyl-CoA trilithium salt. 3H,O and dithiothreitol (DTT) were obtained from Calbiochem and phenylmethylsulfonyl fluoride (PMSF) from Sigma. Penicillin N (PenN), deacetoxycephalosporin C (DAOC), deacetylcephalosporin C (DAC) and cephalosporin C (CephC) were supplied by Ciba-Geigy AG. Sephadex G-100 was purchased from Pharmacia Fine Chemicals and DEAE-Trisacryl M from LKB. All other chemicals were of analytical grade. Organism, media and cultivation
A detailed description of media and conditions used to grow the high yielding strain TR 87 of C. acremonium can be found elsewhere (Scheidegger et al., 1984). Briefly, this involved inoculation of well-grown agar slant cultures into vegetative media and transfer of the 4-day-old vegetative culture into 7.4 1 of solid-free production medium ZEN 1. This culture broth was grown in a 14 1 laboratory bioreactor by feeding first glucose and then soya oil as the carbon source. Preparation of cell-free extract and enzyme purification
All work with cell-free extracts was done between 4 and 10°C. Mycelium was recovered from the cultivation broth at 89 h by centrifugation and washed twice with
111
cold deionized water. 10 g wet cells were suspended in 20 ml cold buffer I (0.05 M Tris-HCl, pH 7.5/0.01 M DTT/O.OOl M PMSF) and added to 20 g precooled glass beads (diam. 0.45-0.50 mm) in a 90 ml glass tube. Cells were ruptured by vibrating the mixture with an immersed glass piston attached to a Vibromixer (Chemap, Typ E 1) for 1 min (frequency 50 Hz). Cell debris was removed by centrifugation at 15 000 X g for 10 min. The proteins of the supernatant were then precipitated with 55% (NH,),SO,. The precipitate was collected by centrifugation and dissolved in buffer I. The solution was desalted on Sephadex G-25 and stored as extract A at - 60°C. Extract A was adsorbed on to a Sephadex G-100 column (3.2 X 40.0 cm), equilibrated and then eluted with buffer I. The enzymatically active fractions were pooled and precipitated with 80% (NH,),SO,. The precipitate was collected by centrifugation and the proteins were dissolved in buffer I. This enzyme solution was either desalted on Sephadex G-25 and the resultant extract B stored at -60°C or loaded on to Sephadex G-25, equilibrated with buffer II (0.01 M Tris-HCl, pH 8.0/0.01 M DTT/O.OOl M PMSF) and then eluted with the same buffer. The eluate was chromatographed on a column (0.8 X 4.0 cm) of DEAE-Trisacryl M, equilibrated with buffer II. Protein was eluted from the column at a flow rate of 10 ml h-’ with a linear gradient (0.01-0.2 M Tris-HCl in 80 ml) of buffer II. The most active fractions were pooled and concentrated by ultrafiltration through a DDS GR 81 P membrane at 5 bar. Protein peaks were monitored at 280 nm. The isoelectric focusing (IEF) was performed on a cooled Pharmacia flat-bed electro-focusing unit. Extract B was focused in a 5% polyacrylamide gel of 0.5 mm thickness (LKB), containing 3% (w/v) Ampholines, ranging from 3.5 to 9.5 pH units. After focusing for 3 h at 800 V, gel fragments were cut out along the pH gradient and eluted into buffer I for 7 h. This solution was tested for enzyme activity. The gels were stained with Coomassie Brilliant Blue R-250 according to LKB instructions. Assays DAC-acetyltransferase:
1 ml of standard reaction mixture contained 0.05 M Tris-HCl, pH 7.5, 4 mM MgSO,, 2 mM acetyl-CoA, 2 mM DAC and extract A (0.1-0.5 mg ml-’ protein, depending on enzyme activity). The reaction was started by addition of DAC and the mixture incubated at 25°C. After lo-45 min, the time varying according to the enzyme activity, the reaction was stopped by adding ethanol (1: 1). The precipitated proteins were removed by centrifugation and the supernatant analyzed by HPLC, using a Zorbax BP-NH, (DuPont) column (4 X 250 mm) and a solvent system as described by Miller et al. (1981). The ionic strength of the solvent system was increased by adding 3 ml 1-l 2 N HCl and 2 N NaOH to adjust the pH to 2.5. CephC served as standard. One unit of enzyme activity is defined as the amount of enzyme which produces 1 pg CephC per minute. The specific activity is defined as units mg-’ protein. Protein was determined according to Bradford (1976). Bovine serum albumin was used as the standard.
112
Results Enzyme test
The reaction product CephC was analyzed by a modified HPLC system according to Miller et al. (1981). The ionic strength was increased so that the system was able to separate DAOC, DAC and CephC completely. CephC eluted after 9 min of separation time (Fig. 1). Its absorption peak was integrated by a computer program to give directly the amount of enzymatically produced CephC in mg ml-‘. No activity could be detected without enzyme extract or with an extract boiled for 10 min. Incubation of the complete test mixture with a protein concentration of 0.1 mg ml-’ showed linearity of the specific activity during the first 40 min. The resultant product on incubation of DAC with crude and partially purified cell extracts was shown to be identical with CephC in three ways. It was a P-lactam by its sensitivity to j%lactamase P-99 (Konecny and Schneider, 1978; Scheidegger et al., 1984) and the UV spectra obtained by UV-range scanning of the peaks and the retention times on HPLC were identical with that of the reference standard of CephC. Enzyme purification
and stability
An initial purification could be achieved by precipitating the enzyme with 55% (NH,),SO,. The specific activity of DAC-acetyltransferase could be increased 2.2-fold after desalting on Sephadex G-25 without too much loss of the total activity. Further purification by Sephadex G-100 gel chromatography caused a 5.1-fold increase in the specific activity of the pooled fractions. The total activity was reduced by 25.7% during this step. The DAC-acetyltransferase eluted very close to albumin, indicating a molecular weight of about 70 000 + 5 000. A further attempt to purify the enzyme by ion exchange chromatography on DEAE-Trisacryl M resulted in a loss of 98% of activity. The specific DAC-acetyltransferase activity after this purification step was 14.5 times higher than that of the crude extract A (Table 1). Since the total activity after ion-exchange purification was too low to allow further purification by IEF the enzyme extract B obtained after Sephadex G-100 gel 0 minutes
II 0
2
4
20 minutes
6
6 10 12 0 2 4 Time (minuted
6
8 x) 12
Fig. 1. HPLC chromatograms of samples taken at two different reaction times
113 TABLE 1 PARTIAL PURIFICATION ACREMONIUM TR 81
OF DAC-ACETYLTRANSFERASE
(1) Crude extract A (2) Desalted O-55% ammonium sulfate precipitate (3) Pooled fractions of Sephadex G-100 chromatography (4) 0-80x ammonium sulfate precipitate of (3) (5) Pooled fractions of DEAE-Trisacryl M chromatography
FROM CEPHALOSPORIIJM
Total protein (m.9 164.4
Total activity (mU 1995.1
Spec. activity (mu mg-‘)
Purification (fold)
2.61
1
337.1
1955.2
5.8
2.2
91.9
1292.3
13.2
5.1
54.2
861.8
15.9
6.1
1.1
41.6
37.8
14.5
chromatography was used. Focusing at 800 V for 3 h revealed a band at a pH of 4.3 f 0.2, which showed activity for the acetylation reaction. Since the protein concentration and activity of the active fraction were low, no data on yield and degree of purification for this step are shown. It was essential to include 1 mM PMSF and 10 mM DTT in all buffers during the purification procedure. For example, inactivation of enzyme activity in an ammonium sulfate-precipitated crude extract could be kept below 12% for a period of 10 h at 4’C. Inclusion of Mercaptoethanol, EDTA, Tween 80, Triton X-100, BSA, DAC and CephC did not stabilise enzyme activity which actually decreased by 40%
Specific Activity ImU mg-1) 1
0
0.2
0.4
0.6
0.6
Substrate ImM)
Fig. 2. Effect of acetyl-CoA and DAC on DAC-acetyltransferase abtivity. Protein concentration: 0.1 mg ml-‘; reaction time: 20 min; concentration of the constant kept second substrate: 2 mM. 0, DAC varied; A, acetyl-CoA varied.
114
Specilic Activity (mU mg-‘1
./
20 /
\
.
./’
15 ./
\
5 “/ . 01
I 10
0
I I 20 30 Temperature PC)
I 40
.
I 50
Fig. 3. Effect of reaction temperature on DAC-acetyltransferase activity. Protein concentration: 0.1 mg ml-‘; reaction time: 20 min.
in 10 h at 4’C. Freezing the enzyme solution in the presence of 10% sucrose or 50% glycerol reduced inactivation by 40% and 45% respectively. Characterization
Mg2+ in concentrations up to 4 mM did not show any significant effect. The dependency on both acetyl-CoA and DAC is shown in Fig. 2. The K, for acetyl-CoA and DAC was calculated to be 0.1 and 0.04 mM, respectively. The optimal temperature of the reaction was determined to be 35°C (Fig. 3). No significant influence of pH on the enzyme activity could be observed in the range of pH values 5-8 when the activity was measured after 20 min incubation (Fig. 4). The reaction was found to be linear up to 40 min at pH 7.5; however, non-linearity and thus some effect of pH on the initial velocity cannot be excluded at other pH values. Specilic Activity
ImU mg-‘)
20 .\e/.-.-* 1 15
10
5
0 I,, 0
----5
6
,
,
7
8 PH
Fig. 4. Effect of pH on DAC-acetyltransferase time: 20 min.
activity. Protein crmcentration: 0.1 mg ml-‘;
reaction
115 TABLE EFFECT Compound (1 mW
2 OF VARIOUS
CELL
added
Control Pyruvate Glucose 6-phosphate a-Ketoglutarate Citrate Fumarate Oxaloacetate Cysteine Valine a-Aminoadipic acid Ascorbate Nicotinic acid Biotin Folic acid Adenosine Cytosine Acetoacetyl-CoA Choline Glyoxylic acid Malonyl-CoA Acetic acid Acetate Propionate Coenzyme A Benzyl alcohol 2,6-Dihydroxybenzoic Deacetoxycephalosporin Penicillin N
METABOLITES
ON DAC-ACETYLTRANSFERASE Relative ce
acid C
ACI-IVITY
activity
100 63 98 100 100 100 89 95 92 94 89 99 90 91 93 92 100 92 100 95 98 100 100 39 98 73 43 86
To study the regulation of the enzyme in vitro, a number of cell metabolites were added at a concentration of 1 mM to the active test mixture. No activators were found (Table 2). Coenzyme A, as a structural analogue of acetyl-CoA, inhibited the activity strongly (61%). Also the DAC analogues deacetoxycephalosporin C (57%), penicillin N (14%) and 2,6-dihydroxybenzoic acid (27%) proved to be inhibitory. Pyruvate exerted an inhibitory effect on DAC-acetyltransferase activity of 37%. Discussion
The new enzyme test system proved to be simple, rapid and precise. The use of radioactively labeled DAC could be avoided. Since the HPLC analysis took only 12 min the entire enzyme test could be accomplished in 40 min. Results were reproducible within 5%. The DAC-acetyltransferase, as the deacetoxycephalosporin C synthetase/hydroxylase, is a very unstable enzyme in crude as well as in partially purified cell extracts.
116
Despite protection of the enzyme against oxidation and proteolytic destruction by respectively adding DTT and PMSF to the buffers, there was still considerable inactivation. The enzyme was very sensitive to freezing in dilute solutions. This could be overcome by adding sucrose or glycerol. No compounds other than DAC and acetyl-CoA were required as substrates for the acetylation. This also held true when the purest active fraction after IEF was tested. The measured extent of the inhibitory effect of PenN and DAOC was similar to that observed by Liersch et al. (1976). An inhibition of DAC-acetyltransferase by DAOC in vivo seems probable, since larger amounts of intracellular DAOC were measured whereas penicillin N and DAC could not be detected (unpublished results). DAOC might therefore exert a negative control on the enzyme. This could help to explain the high amounts of excreted DAC during cultivation. 2,6-Dihydroxybenzoic acid also inhibited the reaction. This may indicate that the binding site of the substrate DAC is more towards the 6-membered ring system than the aminoadipyl moiety. Coenzyme A as a cofactor exerted a strong inhibition whereas acetate did not. The former may bind to the enzyme and, if charged with an acetyl group to bring it into the correct position for reaction with DAC. Possibly the acetyl group does not come into contact with the enzyme. With the exception of pyruvate, no other metabolite tested had a negative effect on the acetylation activity. To get a better knowledge of the regulation of the biosynthesis of CephC it is not only important to study effects that may alter the enzyme activity, but also essential to follow the activity levels of the known enzymes during the cultivation under different regulative conditions. With this new, rapid enzyme test system the realization of this aim has become much easier.
Acknowledgement
The authors thank J.A.L. Auden for reviewing the manuscript.
References Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Felix, H.R., Ntesch, J. and Wehrli, W. (1980) Investigation of the two final steps in the biosynthesis of cephalosporin C using permeabilized cells of Cephalosporium acremonium. FEMS Microbioi. Letters 8, 55-58. Fujisawa, Y. and Kanxaki T. (1975) Role of acetylcoenzyme A: DAC acetyltransferase in cephalosporin C biosynthesis by Cephalosporium acremonium. Agr. Biol. Chem. 39, 2043-2048. Huber, F.M., Baltx, R.H. and Caltrider, P.G. (1968) Formation of deacetylcephalosporin C in cephalosporin C fermentation. Appl. Microbial. 16, 1011-1014. Konecny, J. and Schneider, A. (1978) Alkalimetric microassay of cephalosporins. J. Antibiot. 31. 776-782.
117 Liersch, M., Nliesch J. and TreichIer, H.J. (1976) Final steps in the biosynthesis of cephalosporin C. In: Proc. 2nd Int. Symp. Genet. Ind. Microorg. (K.D. Macdonald, ed.), 74, 179-195. Academic Press, London. Miller, R.D., Huckstep, L.L., McDermott, J.P., Queener, S.W., Kukolja. S., Spry, D.O., Elzey, T.K., Lawrence, SM. and Neuss, N. (1981) High performance liquid chromatography (HPLC) of natural products. IV. The use of HPLC in biosynthetic studies of cephalosporin C in the cell-free system. J. Antibiot. 34, 984-993. Scheidegger, A., Ktenzi, M.T. and Niiesch, J. (1984) Partial purification and catalytic properties of a bifunctional enzyme in the biosynthetic pathway of p-lactams in Cephalosporium acremonium. J. Antibiot. 37, 522-531.
of Biotechnology,
3 (1985)
109
109-117
Elsevier JBT 00171
Investigatioin.of acetyl-CoA: Deacetylcephalosporin C O-acetyltransferase of Cephalosporium acremonium Alfred
Scheidegger ‘** Alois Gutzwiller r, Martin Armin Fikhter 2 and Jakob Niiesch’
’ Research Laboratories .’ Institute for
of the Pharmaceutical Division of Ciba - Geigy Ltd., Biotechnology, ETH - Hiinggerberg, CH - 8093 Zurich,
T. Kienzi
‘,
CH - 4002 Baste, Switzerland
and
(Received 3 April 1985; accepted 22 June 1985)
A new, rapid test system to measure the activity of acetyl-CoA : deacetylcephalosporin C 0-acetyltransferase (DAC-acetyltransferase) was established. The ieaction product cephalosporin C could be easily analyzed by HPLC. The DAC-acetyltransferase was partially purified by means of fractionated (NH&SO, precipitation, Sephadex G-100 gel chromatography and isoelectric focusing. Molecular weight was determined to be 70 000 f 5 000 and the ~14.3 f 0.2. Besides the two substrates acetyl-CoA and deacetylcephalosporin C, no other factor necessary for the reaction could be found. The enzyme was inhibited by coenzyme A (61%), deacetoxycephalosporin C (57%), penicillin N (14%), 2,6dihydroxybenzoic acid (27%) and pyruvate (37%) at a concentration of 0.1 mM in each case. Cephalosporium
acremonium,
deacetylcephalosporin
C, acetyltransferase,
enzyme
characterization
Introduction
One major problem
of the bioproduction of cephalosporin C (CephC) with is the accumulation of considerable amounts of the precursor deacetylcephalosporin C (DAC) during the later part of the cultivation Cephalosporium acremonium
* Present
address:
0168-1656/85/$03.30
Institute for Chemical Research, Kyoto University, Uji, Kyoto-fu 611, Japan. 0 1985 Elsevier Science Publishers B.V. (Biomedical Division)
110
(Huber et al., 1968). The excreted DAC leads to a loss in the CephC yield. DAC is formed either via the biosynthetic route from deacetoxycephalosporin C (DAOC) or by enzymatic and chemical hydrolyzation of CephC. Under well-controlled cultivation conditions high yielding strains will only release low concentrations of esterase activity. Thus, the large amounts of DAC found are most probably the result of an incomplete biosynthesis of CephC. To be able to control positively the acetylation reaction, knowledge of the enzyme involved is a prerequisite. The presence of the catalysing enzyme acetyl-CoA : DAC o-acetyltransferase (DAC-acetyltransferase) was demonstrated in cell free extracts as well as in permeabilized cells by measuring the rate of incorporation of [1-‘4C]acetyl-CoA into CephC (Fujisawa and Kanzaki, 1975; Liersch et al., 1976; Felix et al., 1980). Either acetyl-CoA or acetate plus coenzyme A and ATP served as the acetyl donor. Fujisawa and Kanzaki (1975) described the enzyme to be dependent on Mg2+ as a cofactor, whereas Liersch et al. (1976) could not find such a dependency. The enzyme test method used by these authors has the disadvantage of being time consuming and limited in accuracy and determinations of kinetic data and a characterization of the enzyme are hardly possible. To investigate the properties of the enzyme and in order to obtain a more detailed understanding of the regulation of the terminal reaction in the biosynthetic pathway of CephC, a more precise and rapid enzyme test system is required. This paper deals with a new system for measuring DAC-acetyltransferase activity, the partial purification of this enzyme and its characterization and regulation.
Materials and Methods Materials
Acetyl-CoA trilithium salt. 3H,O and dithiothreitol (DTT) were obtained from Calbiochem and phenylmethylsulfonyl fluoride (PMSF) from Sigma. Penicillin N (PenN), deacetoxycephalosporin C (DAOC), deacetylcephalosporin C (DAC) and cephalosporin C (CephC) were supplied by Ciba-Geigy AG. Sephadex G-100 was purchased from Pharmacia Fine Chemicals and DEAE-Trisacryl M from LKB. All other chemicals were of analytical grade. Organism, media and cultivation
A detailed description of media and conditions used to grow the high yielding strain TR 87 of C. acremonium can be found elsewhere (Scheidegger et al., 1984). Briefly, this involved inoculation of well-grown agar slant cultures into vegetative media and transfer of the 4-day-old vegetative culture into 7.4 1 of solid-free production medium ZEN 1. This culture broth was grown in a 14 1 laboratory bioreactor by feeding first glucose and then soya oil as the carbon source. Preparation of cell-free extract and enzyme purification
All work with cell-free extracts was done between 4 and 10°C. Mycelium was recovered from the cultivation broth at 89 h by centrifugation and washed twice with
111
cold deionized water. 10 g wet cells were suspended in 20 ml cold buffer I (0.05 M Tris-HCl, pH 7.5/0.01 M DTT/O.OOl M PMSF) and added to 20 g precooled glass beads (diam. 0.45-0.50 mm) in a 90 ml glass tube. Cells were ruptured by vibrating the mixture with an immersed glass piston attached to a Vibromixer (Chemap, Typ E 1) for 1 min (frequency 50 Hz). Cell debris was removed by centrifugation at 15 000 X g for 10 min. The proteins of the supernatant were then precipitated with 55% (NH,),SO,. The precipitate was collected by centrifugation and dissolved in buffer I. The solution was desalted on Sephadex G-25 and stored as extract A at - 60°C. Extract A was adsorbed on to a Sephadex G-100 column (3.2 X 40.0 cm), equilibrated and then eluted with buffer I. The enzymatically active fractions were pooled and precipitated with 80% (NH,),SO,. The precipitate was collected by centrifugation and the proteins were dissolved in buffer I. This enzyme solution was either desalted on Sephadex G-25 and the resultant extract B stored at -60°C or loaded on to Sephadex G-25, equilibrated with buffer II (0.01 M Tris-HCl, pH 8.0/0.01 M DTT/O.OOl M PMSF) and then eluted with the same buffer. The eluate was chromatographed on a column (0.8 X 4.0 cm) of DEAE-Trisacryl M, equilibrated with buffer II. Protein was eluted from the column at a flow rate of 10 ml h-’ with a linear gradient (0.01-0.2 M Tris-HCl in 80 ml) of buffer II. The most active fractions were pooled and concentrated by ultrafiltration through a DDS GR 81 P membrane at 5 bar. Protein peaks were monitored at 280 nm. The isoelectric focusing (IEF) was performed on a cooled Pharmacia flat-bed electro-focusing unit. Extract B was focused in a 5% polyacrylamide gel of 0.5 mm thickness (LKB), containing 3% (w/v) Ampholines, ranging from 3.5 to 9.5 pH units. After focusing for 3 h at 800 V, gel fragments were cut out along the pH gradient and eluted into buffer I for 7 h. This solution was tested for enzyme activity. The gels were stained with Coomassie Brilliant Blue R-250 according to LKB instructions. Assays DAC-acetyltransferase:
1 ml of standard reaction mixture contained 0.05 M Tris-HCl, pH 7.5, 4 mM MgSO,, 2 mM acetyl-CoA, 2 mM DAC and extract A (0.1-0.5 mg ml-’ protein, depending on enzyme activity). The reaction was started by addition of DAC and the mixture incubated at 25°C. After lo-45 min, the time varying according to the enzyme activity, the reaction was stopped by adding ethanol (1: 1). The precipitated proteins were removed by centrifugation and the supernatant analyzed by HPLC, using a Zorbax BP-NH, (DuPont) column (4 X 250 mm) and a solvent system as described by Miller et al. (1981). The ionic strength of the solvent system was increased by adding 3 ml 1-l 2 N HCl and 2 N NaOH to adjust the pH to 2.5. CephC served as standard. One unit of enzyme activity is defined as the amount of enzyme which produces 1 pg CephC per minute. The specific activity is defined as units mg-’ protein. Protein was determined according to Bradford (1976). Bovine serum albumin was used as the standard.
112
Results Enzyme test
The reaction product CephC was analyzed by a modified HPLC system according to Miller et al. (1981). The ionic strength was increased so that the system was able to separate DAOC, DAC and CephC completely. CephC eluted after 9 min of separation time (Fig. 1). Its absorption peak was integrated by a computer program to give directly the amount of enzymatically produced CephC in mg ml-‘. No activity could be detected without enzyme extract or with an extract boiled for 10 min. Incubation of the complete test mixture with a protein concentration of 0.1 mg ml-’ showed linearity of the specific activity during the first 40 min. The resultant product on incubation of DAC with crude and partially purified cell extracts was shown to be identical with CephC in three ways. It was a P-lactam by its sensitivity to j%lactamase P-99 (Konecny and Schneider, 1978; Scheidegger et al., 1984) and the UV spectra obtained by UV-range scanning of the peaks and the retention times on HPLC were identical with that of the reference standard of CephC. Enzyme purification
and stability
An initial purification could be achieved by precipitating the enzyme with 55% (NH,),SO,. The specific activity of DAC-acetyltransferase could be increased 2.2-fold after desalting on Sephadex G-25 without too much loss of the total activity. Further purification by Sephadex G-100 gel chromatography caused a 5.1-fold increase in the specific activity of the pooled fractions. The total activity was reduced by 25.7% during this step. The DAC-acetyltransferase eluted very close to albumin, indicating a molecular weight of about 70 000 + 5 000. A further attempt to purify the enzyme by ion exchange chromatography on DEAE-Trisacryl M resulted in a loss of 98% of activity. The specific DAC-acetyltransferase activity after this purification step was 14.5 times higher than that of the crude extract A (Table 1). Since the total activity after ion-exchange purification was too low to allow further purification by IEF the enzyme extract B obtained after Sephadex G-100 gel 0 minutes
II 0
2
4
20 minutes
6
6 10 12 0 2 4 Time (minuted
6
8 x) 12
Fig. 1. HPLC chromatograms of samples taken at two different reaction times
113 TABLE 1 PARTIAL PURIFICATION ACREMONIUM TR 81
OF DAC-ACETYLTRANSFERASE
(1) Crude extract A (2) Desalted O-55% ammonium sulfate precipitate (3) Pooled fractions of Sephadex G-100 chromatography (4) 0-80x ammonium sulfate precipitate of (3) (5) Pooled fractions of DEAE-Trisacryl M chromatography
FROM CEPHALOSPORIIJM
Total protein (m.9 164.4
Total activity (mU 1995.1
Spec. activity (mu mg-‘)
Purification (fold)
2.61
1
337.1
1955.2
5.8
2.2
91.9
1292.3
13.2
5.1
54.2
861.8
15.9
6.1
1.1
41.6
37.8
14.5
chromatography was used. Focusing at 800 V for 3 h revealed a band at a pH of 4.3 f 0.2, which showed activity for the acetylation reaction. Since the protein concentration and activity of the active fraction were low, no data on yield and degree of purification for this step are shown. It was essential to include 1 mM PMSF and 10 mM DTT in all buffers during the purification procedure. For example, inactivation of enzyme activity in an ammonium sulfate-precipitated crude extract could be kept below 12% for a period of 10 h at 4’C. Inclusion of Mercaptoethanol, EDTA, Tween 80, Triton X-100, BSA, DAC and CephC did not stabilise enzyme activity which actually decreased by 40%
Specific Activity ImU mg-1) 1
0
0.2
0.4
0.6
0.6
Substrate ImM)
Fig. 2. Effect of acetyl-CoA and DAC on DAC-acetyltransferase abtivity. Protein concentration: 0.1 mg ml-‘; reaction time: 20 min; concentration of the constant kept second substrate: 2 mM. 0, DAC varied; A, acetyl-CoA varied.
114
Specilic Activity (mU mg-‘1
./
20 /
\
.
./’
15 ./
\
5 “/ . 01
I 10
0
I I 20 30 Temperature PC)
I 40
.
I 50
Fig. 3. Effect of reaction temperature on DAC-acetyltransferase activity. Protein concentration: 0.1 mg ml-‘; reaction time: 20 min.
in 10 h at 4’C. Freezing the enzyme solution in the presence of 10% sucrose or 50% glycerol reduced inactivation by 40% and 45% respectively. Characterization
Mg2+ in concentrations up to 4 mM did not show any significant effect. The dependency on both acetyl-CoA and DAC is shown in Fig. 2. The K, for acetyl-CoA and DAC was calculated to be 0.1 and 0.04 mM, respectively. The optimal temperature of the reaction was determined to be 35°C (Fig. 3). No significant influence of pH on the enzyme activity could be observed in the range of pH values 5-8 when the activity was measured after 20 min incubation (Fig. 4). The reaction was found to be linear up to 40 min at pH 7.5; however, non-linearity and thus some effect of pH on the initial velocity cannot be excluded at other pH values. Specilic Activity
ImU mg-‘)
20 .\e/.-.-* 1 15
10
5
0 I,, 0
----5
6
,
,
7
8 PH
Fig. 4. Effect of pH on DAC-acetyltransferase time: 20 min.
activity. Protein crmcentration: 0.1 mg ml-‘;
reaction
115 TABLE EFFECT Compound (1 mW
2 OF VARIOUS
CELL
added
Control Pyruvate Glucose 6-phosphate a-Ketoglutarate Citrate Fumarate Oxaloacetate Cysteine Valine a-Aminoadipic acid Ascorbate Nicotinic acid Biotin Folic acid Adenosine Cytosine Acetoacetyl-CoA Choline Glyoxylic acid Malonyl-CoA Acetic acid Acetate Propionate Coenzyme A Benzyl alcohol 2,6-Dihydroxybenzoic Deacetoxycephalosporin Penicillin N
METABOLITES
ON DAC-ACETYLTRANSFERASE Relative ce
acid C
ACI-IVITY
activity
100 63 98 100 100 100 89 95 92 94 89 99 90 91 93 92 100 92 100 95 98 100 100 39 98 73 43 86
To study the regulation of the enzyme in vitro, a number of cell metabolites were added at a concentration of 1 mM to the active test mixture. No activators were found (Table 2). Coenzyme A, as a structural analogue of acetyl-CoA, inhibited the activity strongly (61%). Also the DAC analogues deacetoxycephalosporin C (57%), penicillin N (14%) and 2,6-dihydroxybenzoic acid (27%) proved to be inhibitory. Pyruvate exerted an inhibitory effect on DAC-acetyltransferase activity of 37%. Discussion
The new enzyme test system proved to be simple, rapid and precise. The use of radioactively labeled DAC could be avoided. Since the HPLC analysis took only 12 min the entire enzyme test could be accomplished in 40 min. Results were reproducible within 5%. The DAC-acetyltransferase, as the deacetoxycephalosporin C synthetase/hydroxylase, is a very unstable enzyme in crude as well as in partially purified cell extracts.
116
Despite protection of the enzyme against oxidation and proteolytic destruction by respectively adding DTT and PMSF to the buffers, there was still considerable inactivation. The enzyme was very sensitive to freezing in dilute solutions. This could be overcome by adding sucrose or glycerol. No compounds other than DAC and acetyl-CoA were required as substrates for the acetylation. This also held true when the purest active fraction after IEF was tested. The measured extent of the inhibitory effect of PenN and DAOC was similar to that observed by Liersch et al. (1976). An inhibition of DAC-acetyltransferase by DAOC in vivo seems probable, since larger amounts of intracellular DAOC were measured whereas penicillin N and DAC could not be detected (unpublished results). DAOC might therefore exert a negative control on the enzyme. This could help to explain the high amounts of excreted DAC during cultivation. 2,6-Dihydroxybenzoic acid also inhibited the reaction. This may indicate that the binding site of the substrate DAC is more towards the 6-membered ring system than the aminoadipyl moiety. Coenzyme A as a cofactor exerted a strong inhibition whereas acetate did not. The former may bind to the enzyme and, if charged with an acetyl group to bring it into the correct position for reaction with DAC. Possibly the acetyl group does not come into contact with the enzyme. With the exception of pyruvate, no other metabolite tested had a negative effect on the acetylation activity. To get a better knowledge of the regulation of the biosynthesis of CephC it is not only important to study effects that may alter the enzyme activity, but also essential to follow the activity levels of the known enzymes during the cultivation under different regulative conditions. With this new, rapid enzyme test system the realization of this aim has become much easier.
Acknowledgement
The authors thank J.A.L. Auden for reviewing the manuscript.
References Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Felix, H.R., Ntesch, J. and Wehrli, W. (1980) Investigation of the two final steps in the biosynthesis of cephalosporin C using permeabilized cells of Cephalosporium acremonium. FEMS Microbioi. Letters 8, 55-58. Fujisawa, Y. and Kanxaki T. (1975) Role of acetylcoenzyme A: DAC acetyltransferase in cephalosporin C biosynthesis by Cephalosporium acremonium. Agr. Biol. Chem. 39, 2043-2048. Huber, F.M., Baltx, R.H. and Caltrider, P.G. (1968) Formation of deacetylcephalosporin C in cephalosporin C fermentation. Appl. Microbial. 16, 1011-1014. Konecny, J. and Schneider, A. (1978) Alkalimetric microassay of cephalosporins. J. Antibiot. 31. 776-782.
117 Liersch, M., Nliesch J. and TreichIer, H.J. (1976) Final steps in the biosynthesis of cephalosporin C. In: Proc. 2nd Int. Symp. Genet. Ind. Microorg. (K.D. Macdonald, ed.), 74, 179-195. Academic Press, London. Miller, R.D., Huckstep, L.L., McDermott, J.P., Queener, S.W., Kukolja. S., Spry, D.O., Elzey, T.K., Lawrence, SM. and Neuss, N. (1981) High performance liquid chromatography (HPLC) of natural products. IV. The use of HPLC in biosynthetic studies of cephalosporin C in the cell-free system. J. Antibiot. 34, 984-993. Scheidegger, A., Ktenzi, M.T. and Niiesch, J. (1984) Partial purification and catalytic properties of a bifunctional enzyme in the biosynthetic pathway of p-lactams in Cephalosporium acremonium. J. Antibiot. 37, 522-531.