Eur. J. Biochem. 58. 467-475 (1975)

Patulin Biosynthesis : The Metabolism of m-Hydroxybenzyl Alcohol and m-Hydroxybenzaldehyde by Particulate Preparations from Penicillium patulum Gilhdn MURPHY and Feodor LYNEN

Max-Pkanck-Institut fur Biochemie, Martinsried

(Received April 7,’July 4, 1975)

The ring hydroxylation of m-hydroxybenzyl alcohol to gentisyl alcohol by a particulate prepara- tion from Penicilliumpatulum has been characterised. The activity was shown to be closely associated with, but not necessarily identical to, m-cresol 2-hydroxylase activity of the 105000 x g microsomal fraction. As with both the m-cresol hydroxylases of this system, m-hydroxybenzyl alcohol hydroxylase requires oxygen and NADPH for activity. A K , value for m-hydroxybenzyl alcohol of 15 pM was measured. Inhibition of the hydroxylase activity and its reversal by light, as well as the action of cyto- chrome c, KCN and other effectors suggested a mixed-function oxidase reaction of the cytochrome P-450, NADPH-cytochrome reductase type.

m-Hydroxybenzaldehyde was not ring hydroxylated by any preparation from P. patulum. Apart from the previously described conversion to m-hydroxybenzyl alcohol by a predominantly soluble dehydrogenase, m-hydroxybenzaldehyde was metabolized to m-hydroxybenzoic acid by a particulate fraction. This activity required NADPH.

It was concluded that the main biosynthetic pathway to patulin must be through m-hydroxybenzyl alcohol, gentisyl alcohol and gentisaldehyde.

Radioactive tracer experiments involving the use of labelled metabolites have provided evidence for their involvement in the biosynthetic pathway from 6-methylsalicylic acid to patulin in Penicillium patulum cultures and have given some indication of the inter- relationship between the metabolites observed during secondary metabolism of this fungus [1,2]. The demonstration of these conversions at the enzyme lcvel in cell-free systems has been more difficult, but in recent years a number of soluble enzyme activities involved in patulin biosynthesis have been characteris- ed (Fig. 4). 6-Methylsdlicyclic acid decarboxylase has been fully purified and characterised by Vogel [3] in this laboratory and Forrester and Gaucher [4] have dcscribed the partial purification of an alcohol dehy- drogenase specific for the interconversion of m- hydroxybenzyl alcohol and m-hydroxybenzaldehydc. Recently, Scott and Beadling [5] isolated and partially purified two inseparable dehydrogenase activities

Enzymes. m-Hydroxybenzyl alcohol 2-hydroxylase (m-hydroxy- beniyl alcohol, reduced NADP : oxygen oxidoreductase), rn-cresol 1 -methylhydroxylase and m-cresol 2-hydroxykase (m-cresol, re- duced NADP: oxygen oxidoreductases) (EC 1.14.13.-).

catalysing the reversible conversions of m-hydroxy- benzyl alcohol and gentisyl alcohol to their correspond- ing aldehydes. They also described a 10OOOOxg supernatant activity catalysing the conversion of gentisaldehyde to patulin. We have reported two particulate hydroxylase activities, converting rn-cresol to 2,5-dihydroxytoluene or to m-hydroxybenzyl alco- hol, the latter being significant in the synthesis of patulin [6]. This study describes the further metab- olism of m-hydroxybenzyl alcohol and m-hydroxy- benzaldehyde by particulate fractions from P. patulum to complete the enzymic characterisation of the main patulin biosynthetic sequence (Fig. 4).

MATERIALS AND METHODS Materiuls

General materials, radiochemicals and P . patulum cultures were obtained from the sources given in Murphy et al. [6]. m-Hydroxybenzaldehyde, m-hy- droxybenzoic acid and gentisaldehyde were from Th. Schuchardt (Munchen) and 6,7-dirnethyltetrahy- dropterin was the generous gift of Dr G. Hennings.

468 Microsomal Activities in Patulin Biosynthesis

M e tlzocls

Cultivation of Penicillium patulum CBS 384.48 was carried out as described in Murphy et al. [6]. The majority of preparations dcscribed were taken from mycel after 24h of submerged fermentor culture, rather than after a 48-h period, as used previously. Mycel breakdown has also been dcscribcd; supcr- natant preparations were made at 10000 x g for 10 min, as this routinely gave 100% recovery of m-hydroxy- bcnzyl alcohol hydroxykasc activity from raw extracts. The protein content was 14- 19 mg/ml. The micro- soma1 fraction was prepared by centrifugation of the 10000 x g supernatant at 105000 x g for 30 min and resuspension of the pellet in buffer to 0.8 times the volume of the starting material. This had a protein content of 9.5 - 11 mg/ml. "Washed" microsomes were prepared by resuspension of such pellet material in twice the volume of buffer and recentrifugation as above.

The procedure for sucrose density gradient centri- fugation in 15 - 35 %, (w/v) sucrose has been described @I.

Gradients of 9 ml 10-40';/, (w/v) sucrosc, layered onto 1 ml of 50 (w/v) and 1 ml of 60 %, (w/v) sucrose were also used, with a 3-ml sample of 10000 x g mycel supernatant. In this case centrifugation was for 30 min at 200000 x g .

Preparation of m-Hydro~y('~C]benzyl Alcohol

Mycel(600 mg), cultivatcd and washed as described previously, was incubated in 20 ml of 100 mM potas- sium phosphate buffer, pH 6.5, containing 1 mM sodium [14C]-acetate (10' counts/min) and 1 mM m-hydroxybenzyl alcohol at 30 "C for 1-2 h. The medium and mycel washings were extracted, dried and concentrated prior to thin-layer chromatography ( x 2) in chloroform/ethylacetate/ether (5/2/1, by vol.) [6]. The region corresponding to m-hydroxybenzyl alcohol was located by ultraviolet fluorescence and radioactive scanning, scraped off and eluted with ether/methanol(9/1, v/v). The eluate was concentrated and re-chromatographed ( x 2) on silica gel-kieselgur (0.25 mm thick, from E. Merck, Darmstadt) in chloro- form/ethyl acetate/acetic acid (70/28/2, by vol. ; Table l), with final detection and elution of the m- hydroxybenzyl alcohol band as before. The resultant material gave a symmetrical peak of radioactivity on thin-layer chromatography in a number of solvent systems, with a mobility corresponding to authentic m-hydroxybenzyl alcohol (Table 1). Trimethylsilyla- tion and gas-liquid chromatography [6] indicated a single radioactive product, identical to m-hydroxy- benzyl alcohol. Addition of a known amount (79 600 counts/min) of the radioactive material to 100 mg unlabelled, authentic m-hydroxybenzyl alcohol

and repeated recrystallisation with 1,2-dichloroethane gavc a product of specific radioactivity 780-815 counts x min-' x mg-', with a melting point of 67 "C (67 "C; [7]). The concentration of radioactive prepara- tions was estimated using the ultraviolet absorption data of Forrester and Gaucher [l].

Preparation of m-Hydro.~y('~C]benzaldehyde

rn-Hydr~xy['~C]benzyl alcohol, prepared as de- scribed above, was mildly oxidised using 17; (w/v) chromic anhydride in pyridine [S]. A washed ether cxtract of the ethanol-quenched, acidified reaction mixture was chromatographed on silica gel-kieselgur in chloroform/ethyl acetate/acetic acid (70/28/2, by vol.). The radioactive, fluorescing band corresponding to authentic m-hydroxybcnzaldchydc was scraped off and eluted. It was found to have identical mobility to authentic material in a number of thin-layer chromatographic systems (Table 1). Addition of a known amount of the rn-hydro~y['~C]benzaldehyde to unlabclled material (initial specific radioactivity 1160 counts x min-' x mg-') and repeated recrystal- lisation rrom benzene gave a product of specific radioactivity 1130- 1150 counts x min-' x mg-'. mclting point 104 "C (106 " C ; [9]). The concentration of radioactive preparations was adjusted using the ultraviolet absorption data of Forrester and Gaucher [I]. 80% conversion of the alcohol to the aldehyde was obtainable by this method.

Prepration of Gentisyl Alcohol

Gentisyl alcohol was prepared by reduction of gentisaldehyde using sodium borohydride, as described by Scott et ul. [2]. Initial purification by two-fold thin- layer chromatography on silica gel-kieselgur in chlo- roform/ethyl acetate/acetic acid (70/28/2, by vol.) was carried out, followed by recrystallisation from chloro- form/methanol. The melting point of the final material was 101 "C (99- 100 "C; [lo]).

Assay of Enzyme Activities

m-Cresol hydroxylases were assayed as described previously [6]. The assay for m-hydroxybenzyl alcohol hydroxylase activity was basically similar, employing 0.4 mM m-hydro~y['~C]benzyl alcohol (1.5 x 105-2.0 x lo5 counts x min-' x pmol-') as substrate in an incubation of final volume 1 ml. Blank values, taken to be the incorporation of thc substrate into gentisyl alcohol in the absence of NADPH, were higher and more variable (200- 300 counts/min) than for the m-cresol hydroxylases. Generally, assays were carried out using a 50 p1 volume of 10000 x g supernatant or microsomal fraction, with a 5-min incubation period at 25 "C (Fig. 1). The majority of observations de-

G. Murphy and F. Lynen 469

Table 1. Tliv tmbilirj of secorzrlury nietuholites of' P. patulum in various thin-layer clirornatogruphic systems Authentic samplcs of metabolites involved in the biosynthesis of patulin were chromatographed twice over a distance of 17.8 cm on silica gel, S. thin-layer plates or silica gel kieselgur, SK, plates in the solvent system described. R, values for thc system ch1oroform;cthyl acetate/cther. 5i2,l (by vol.) were given in Murphy rt al. [6]

Compound Distance run with system

A B C D E F

cm

wHydroxybenzyl alcohol Gentisyl alcohol ni-Hydroxybenzaldehyde Gentisaldehyde ni-Hydroxybcnzaldehyde-2,4-dinitrophenylhydrazone Gentisaldehydc-2,4-dinitrophcnyl hydrazone ni-Hydroxybenzoic acid Patulin

~ ~~

9 7 11.7 7.3 4.2 3.5 6.2 5.6 5.4 5.0 1.6

14.9 11.9 10.4 12.6 14.1 10.9 10.8 9.1 11.9

11.0 14.1 7.8 11.5

10.9 9.0 2.2 7.8 11.1 8.8 5.6

System ~

by vol. ~ _ _ _ _ _ _ _ _ ______

A Chloroform,ethyl acetatelacetic acid B Chloroform/acetic acid c Benzene dioxan dcetic acid D Chloroform/acctic acid E Toluene, chloroform,acetone F E containing 2",, (v'v) acetic acid

70/28;'2 4,'l 90,2514 9.1 7.2,'l

SK SK S S SK SK

scribed were made using microsomal preparations, but little difference was found between effects on the microsomes and on 10000 x g supernatant prepara- tions. Reactions were stopped by acidification to pH 3 and 1.0 pmol of carrier gentisyl alcohol was added to each incubation, prior to extraction with 4 x 3 ml peroxide-free ether. Extracts were spotted onto silica gel-kieselgur plates and developed twice in the system chloroform/ethyl acetate/acetic acid (70/28/2, by vol.). A better separation of substrate and product was obtainable with the system chloroform/ acetic acid (4/1, v/v), but, due to streaking of brown lipid material extracted from the mycel, visualisation of the developcd plate was not easy (Table 1). The region corresponding to gentisyl alcohol was detected by its ultraviolet fluorescence and was scraped off and counted [6].

Experiments involving a defined gas phase were carried out using Warburg manometers, as described before [6]. In incubations in the absence of oxygen the inclusion of alkaline pyrogallol in the flask ccntre wcll was found to be unnecessary. Light expcriments were also set up as described before, employing a 250-watt lamp projected through a 3-cm layer of 1 (wlv) copper sulphate.

Incubations involving pteridine derivatives were carried out in red light to slow down their decomposi- tion.

The metabolism of m-hydr~xy['~C]benzaldehyde by P. patulum extracts was studied using a 1-ml assay system with 0.2 or 0.4 mM m-hydroxy[14C]- benzaldehyde (3.0 x 10s-400 x dis. x rnin-'

xpmol-') and 0.5 mM NADPH (blanks without NADPH). Supernatant preparations (50 pl) and un- washed microsomal preparations (50 pl) were used in 5-min assays at 25 "C. Incubations were stopped by acidification to pH 3, carrier gentisaldehyde (0.2 pmol), m-hydroxybenzoic acid (1 .O pmol) or m-hydroxybenzyl alcohol (1.5 pmol) were added, as required, and they were saturated with NaCl prior to 3x3-ml ether extraction. For the examination of incorporation into gentisaldehyde, which is difficult to scparate cleanly from m-hydroxybenzaldehyde, extracts wcre spotted onto silica gel-kiesclgur plates and a saturated solution of 2,4-dinitrophenylhydrazine in 2 N HCI ( 5 x 10 PI) was spotted on top of each sample [ll]. After 15-20 min the plates were developed twice in toluene/chloroform/acetone (7/2/1, by vol.) alone, or with the addition of 2 % (v/v) acetic acid (Table 1). The latter served to increase the mobility and decrease the trailing of m-hydroxybenzoic acid and was used in its identification as a product of the incubations. Material corresponding to gentisaldehyde 2,4-dinitro- phenylhydrazone, visible as a brownish-ycllow spot, was scraped off and counted as described previously. Duc to the strong quenching imparted by yellow solutions, corrections of radioactivity wcre made using thc external standard method. For the idcntifica- tion of m-hydroxybenzyl alcohol and m-hydroxy- benzoic acid and the measurcrnent of incorporation into m-hydroxybenzoic acid, extracts werc chro- matographed twice on silica gel-kieselgur in chloro- form/ethyl acetate/acetic acid (70/28/2, by vol.) or on silica gel in chloroform/acetic acid (9/l, v/v; Table 1).

470 Microsomal Activities in Patulin Biosynthesis

Fig. 1 . The eij%ct of time and m:yme concentration on the incorporuiion of rn-hydro.~y('~C] ulcohol into gentisyl alcoliol hy preparurions of'P.patulum. (A) Incubations were carried out for varying lengths of time using a 1 O O O O x g supernatant preparation in (0) air and (0) an atmosphere of 1007; oxygen. (B) Incubations were for 5 min using dilutions of (0) a IOOOOxg supernatant fraction, 16.3 mg proteinlml and (0) a microsomal prcparation, 11.7 mg proteinlml

General identification involved the comparison of radioactive peaks located by scanning with the fluores- cent spot of both radioactive and unlabelled authentic material. Specific incorporation was measured by scraping off the relevant spots and couting.

For the further identification of one of the products of m-hydr~xy['~C]benzaldehyde metabolism as m- hydroxybenzoic acid, the latter was recrystallised as the methyl ester. Eight incubations of m-hydroxy- [14C]benzaldehyde with a washed microsomal prepara- tion were carried out under the conditions described above, in the presence of NADPH, extracted and sub- jected to thin-layer chromatography in chloroform/ acetic acid (9/1, v/v). The radioactive region corre- sponding to m-hydroxybenzoic acid was scraped off and eluted with ether/methanol. After measurement of the total radioactive content, the extract was dried and 100 mg of authentic m-hydroxybenzoic acid was added (initial specific radioactivity 171 counts x min-l pmol-I). This was dissolved in 0.4 ml ethanol and a solution of 1.5% (w/v) diazomethane in ether was added dropwise until the yellow colour persisted and was stood for 10 min prior to removal of excess diazomethane with a stream of nitrogen. The reaction mixture was concentrated, spotted onto a silica gel plate and developed twice in benzene/ethyl acetate (9/l , v/v). Scanning indicated that two radioactive products had been formed, the major band corre- sponding to m-hydroxybenzoic acid methyl ester. No unreacted m-hydroxybenzoic acid remained. The major band was scraped off, eluted with ether and dried. Subsequent repeated recrystallisations from hexane/ benzene yielded crystals of specific radioactivity 169- 175 counts x min-' x pmol and a melting point of 70 C (69-71.5 "C; [12]).

RESULTS Characterisation of m-Hyhxybenzyl Alcohol Hydroxylustj

One radioactive product was observed using m- hydroxyl[14C]benzyl alcohol as a substrate in incuba- tions with P.patulum extracts in the presence of NADPH. This was identified as gentisyl alcohol by thin-layer chromatography in several systems (Table 1) and by trimethylsilylation and gas-liquid chromatog- raphy. In each case the mobility of the radioactive compound was identical to that of authentic, unla- belled material. Under the assay conditions described, crude fungal extracts and the centrifugal fractions used in this study a linear production of gentisyl alcohol with respect to both enzyme concentration and time for a period of up to 5 min (Fig. 1).

Using mycel harvested after 24 h of fermentor growth, m-hydroxybenzyl alcohol hydroxylase and m-cresol hydroxylases of similar activities could be prepared. Values of up to 1.0 pmol x min-' x g dry mycel-' (approximately 160 mg 10000 x g supernatant protein) were measured. These activities had dropped after 48 h of fermenter growth to 0.5-0.7 pmol x m i n - ' x g dry mycel-I. This may be due to the ease of breakdown of the 24-h mycel, as compared to that after 48 h of growth. However, it was found that the 24-h mycel extracts were quite stable with respect to both m-hydroxybenzyl alcohol and m-cresol hy- droxylase activities and could be kept at - 30 " C , in the form of concentrated microsomal suspensions, for several days without activity loss. We had previously found very low stability of m-cresol hydroxylases in 48-h mycel extracts [6] which may be due to higher protease levels in older material (J. Friedrich, personal communication), or the effect of increased phenolic

G. Murphy and F. Lyncn 473

935' 15 Sucrose(%) Gradient volurne(rn1)

0.16 I I

E

I -bo4- .. 10 Sucrose(0') Gradient volume( rnl)

60 50

Fig. 2. Sucrose clensity grudienr ccwrr'i/iigurion i?f low-speed super- ~~u/ur i~ , f , .uc t ions~ron~ P. patulum to separate m-hydroxybenzyl alcohol Iij.ilro.xjluse and the IW m-cresol Iiydro.xjluse uctivities. Centrifuga- tion was carried out as described in the Materials and Methods section in (A) a 15 - 35 "/a (w/v) sucrose gradient with a base of 1 ml of 40% sucrose and (B) a 10-400//, sucrose gradient with a base of 1 ml so:.; and 1 ml 607; sucrose. Activities arc expressed as pmol of substrate incorporated per ml of each fraction in 5 min for m-cresol incorporation into (0) 2.5-dihydroxytoluene and (0) m-hydroxybenzyl alcohol and for m-hydroxybenzyl alcohol in- corporation into (A) gentisyl alcohol

secondary metabolite levels which could be harmful to enzymes in a cell-free preparation. As was observed for the rn-cresol hydroxylases [6], the m-hydroxy- benzyl alcohol-hydroxylating activity was located mainly in the microsomal fraction of mycel extracts, sedimenting between 10000 x g and 105000 xg, re- coveries of from 80-100%, of the initial extract activity being obtained. The 105000 x g supernatant generally retained 2- 10% of this activity.

Sucrose density gradient centrifugation of 24 h mycel extract 10000 x g supernatants at 200000 x g for 1 h gave a rather altered pattern for the 2,5-di- hydroxytoluene and m-hydroxybenzyl alcohol-pro- ducing (m-cresol-hydroxylating) activities to those

Table 2. eflecl of' other metabolites of' the patulin biosjnfhetic path- war on the liyrlroxylation of m-hydroxyhenzyl alcohol The in-hydroxybenzyl alcohol hydroxylase activity of mycel micro- soma1 preparations incubated in the presence of compounds shown to be significant in the biosynthesis of patulin is expressed as a pcrccntage of the activity under normal optimal assay conditions. Similar results were obtained using 10000 x g supernatant prepa- rations

Added metabolite Concentration Relative incorporation into gentisyl alcohol

mM 2, None - m-Cresol 0.1

0.5 1 .o

m-H ydroxy benzaldchyde 1 .o 2.5-Dihydroxytoluene 1 .o Gcntisyl alcohol 1 .o Gentisaldehyde 1 .o Patulin 1 .o

100 13 52 40 4

85 96 49 11

observed previously (Fig.2A and [6]). This was probably due to the different breakdown charac- teristics of the mycel after 24 h of fermentor growth, as compared with 48 h. Modification of the sucrose gradient and centrifugation time gave a better distribu- tion of activities throughout the gradient (Fig. 2 B). The gentisyl alcohol-producing activity was found to sediment in close association with the 2,5-dihydroxy- toluene-producing activity in both gradients, suggest- ing that the two enzymes are probably localised in the same membrane. The rn-hydroxybenzyl alcohol-pro- ducing activity occurred in a more slowly sedimenting membrane fraction.

Using microsomal preparations a K, for m-hy- droxybenzyl alcohol of about 15 pM was found for the 2-hydroxylase activity. The effects of other patulin biosynthetic pathway metabolites [l, 21 on the In- hydroxybenzyl alcohol hydroxylase were comparable to those for m-cresol2-hydroxylase (producing 2,5-di- hydroxytoluene), particularly in that m-hydroxybenz- aldehyde showed significant inhibition of the reaction (Table 2). The observation that m-cresol also had a strongly inhibitory action on m-hydroxybenzyl alcohol hydroxylation, whereas rn-hydroxybenzyl alcohol in- hibited m-cresol hydroxylation, suggests that one enzyme may catalyse both reactions as a rather non- specific ring hydroxylase of methyl-substituted phe- nols.

The pH-activity curve of m-hydroxybenzyl alcohol hydroxylase at optimal substrate concentrations corre- sponded closely to that of the rn-cresol hydroxylases (Fig.3). A specific requirement of the activity for NADPH as the reduced cofactor was also found (Table 3), NADH or transhydrogenase activity hav-

412 Microsomal Activities in Patulin Biosyiithesis

PH Fig. 3. pH-activity curws .fiw rn-hytlroxyhenzyl alcohol hydroxjlose from P. patulum. Activities were assayed using a microsomal prep- aration incubated at varying pH values in (.) 100 mM potassium phosphatc buffer or (0) 100 mM Tris-HCI buffer at optimal sub- strate concentrations

Table 3. lirecr of pyridine nucleotides on m - l ~ y d r o s y [ ' ~ C ] h ~ ~ ~ i ~ y l alcolzol hydruxylution b j P. patulum microsomal preparations Activity in the presence of various pyridine nucleotides is expressed rclative to the activity using NADPH under optimal assay con- ditions

Pyridine nucleotide Concentration Relativc incorporation into gentisyl alcohol

o / in M , c,

NADPH 0.5 100 NADH 0.5 6 NADPH + NADH 0.5 124

NADH + ATP 0.5 4

NADH + NADP+ 0.5 8.5

NADH + ATP + NADP+ 0.5 11

2.0

0.5

0.5

0.5 0.5

ing markedly less effect. A slight enhancement of activity was obtained with a mixture of NADPH and NADH, but did not approach the two-fold, synergistic effect found by Duppel et al. [13] in a yeast hydroxylating enzyme system. Oxygen was also found to be necessary for nT-hydroxybenzyl alcohol hydroxyl- ation, no activity being observed in an atmosphere of 100% Nz (Table 4). Saturation of incubations with oxygen (1OO'X Oz atmosphere) gave a small, but significant increase in gentisyl alcohol production, but 100% O2 did not improve the linearity of the assay with respect to time or enzyme concentration (Fig. 1). m-Hydroxybenzyl alcohol hydroxylase activity was generally found to be more sensitive to low oxygen concentrations than the two m-cresol hydroxylases (Tablc 4).

Table 4. The activity oj m-lzydroxybenzjl ulcohol orid m-crrsol hj~drusjbses a1 varying oryzen concentrations Incubations were carried out as described in the Materials and Methods section, using 10000 x g supernatant preparations from P.patulum. Thc cffect of different concentrations of oxygen on the incorporation of m-['4C]cresol into 2,5-dihydroxytolucnc (1) and m-hydroxybcnzyl alcohol (11) and of m-hydr~xy['~C]benzyl alcohol into gentisyl alcohol (111) are cxprcsscd rclative to their activities in air

Gas phase Rclative incorporatioil into

~

o/ ' 0

~

100 0 6 3 2 95 5 91 98 82. 90 10 91 100 89 80 20 99 101 104 0 100 101 99 109

Table 5. Tht oction uf vuriuus rnised+nction o.riciaw c:j@etors on the liydroxylation c?f'm-ltydr(~s?Srn~yl alcohol Supernatant (10000 x g ) (S) or rnicrosomal (M) fractions from P.patulurn were assayed for their ability to incorporate ni-hydroxy- ['4C]bcnzyl alcohol into gentisyl alcohol in the presence ofa nuinbcr of reported effectors of the different types of mixed-function oxidase activity thal have been characteriscd. Thc rcsults arc expressed rclative to the activity under optimal conditions. All the effectors had no activity in the absence of NADPM

Added effector Concen- Relative Frac- tration incorporation tion

into gcntisyl alcohol

None EDTA

(Et),NCS,Na"

Z,Z'-Dipyridyl o-Phenanthroline

6,7-Dimethyl tetra- hydropterin

Tetrahydrofolatc Ascorbate

Catechol

Iodoacetamide Carbon monoxide Cytochroine c KCN Cytochrome c + KCN

niM -

5 10

1 5 1 0.5 3.0

0.1 0.5 0.1 4

10 0.0025 0.05 1 0.9 0.1 1 0.1 1

% 100 106 S 102 S 105 M 82.5 M

103 S 99 S 86 S

80 S 70 S 89.5 S

100 S 91.5 S 91.5 M 86.5 M 98.5 S

3 S 31, 5' M 99,100' M 83. 61' M

a Sodium diethyldithiocarbamate. Saturating concentration. At 0.1 mM NADPH.

G. Murphy and F. Lynen 413

Tablc 6 . Carhon nionoside inhihition of m-hydro.yybenrxl alcohol Iiwlro.vj.lase and iis reversal h j lighr in comparison with the cfject oi i m-crrsol IJjYlI'O.Vj'kfISt3 Thc inhibitory cflcct of carbon moiioxide on the ability of P. Paiirlirni supernatant proparations to hydroxylate rn-cresol. pro- ducing 2.5-dihydroxytoluene ( I ) and ni-hydroxybenzyl alcohol (11) and to hydroxylate ni-hydroxybenzyl alcohol, producing gentisyl alcoliol ( I l l ) was studied. Incubations were sct up as dcscribcd previously [6]. using two ratios of carbon monoxide to oxygen at varying oxygen concentrations. A comparison of the hydroxylase activities in normal light and under strong illumination is cx- prcsscd relative to thc activities in thc prcscncc of thc oxygcn concentration alone

Gas phase Relative incorporation into

Oz CO:Oz dark light

I 11 111 I 11 111

0 0

5 2 . 1 18 22 32 55 51 62 5 1 : l 21.5 33 51 64 56 62

10 2 : l 13.5 21 31 52 41.5 54 10 1 : l 22.5 32 58 51.5 50.5 58.5

- 54 - 29 20 7 : l - 51 20 I : 1 - - - - 40

-

As the hydroxylation reaction once more appeared to be of the mixed-function oxidase type, further characterisation was carried out by examining the action of a variety of effectors known to influence the activity of such enzymes (Table 5). Similar to the rn-cresol hydroxylases, metal ions and metal chelators had no effect and ascorbate, catechol or tetrahydro- pteridine derivatives did not act as reducing cofactors in place of, or complementary to NADPH [6]. At saturating concentration carbon monoxide was com- pletely inhibitory to gentisyl alcohol formation from m-hydroxybenzyl alcohol and significant inhibition by cytochrome c was observed, partially reversible by the addition of the cytochrome oxidase inhibitor, potassium cyanide. Table 6 compares the varying inhibitory effects of two ratios of carbon monoxide to oxygen at three oxygen concentrations on the m- hydroxybenzyl alcohol and m-cresol hydroxylases, and the action of irradiation by strong light of 380- 520 nm in the reversal of this inhibition. rn-Hydroxy- benzyl alcohol hydroxylase was found to be less sensitive to carbon monoxide inhibition and light reactivation than the two m-cresol hydroxylases. This may be a reflection of the non-optimal activity at some of the oxygen concentrations employed. The involvement of an NADPH-dependent cytochrome reductase and of a pigment like cytochrome P-450, responsible for oxygen activation in a large number of mixed-function oxidase reactions in both mammalian and bacterial systems, are implicated by these results. Competition between carbon monoxide and oxygen

for a cytochrome oxygen acceptor is reflected in the similarity of inhibition and reversal of the hydroxylase activities a t a constant carbon monoxide to oxygen ratio, with changing oxygcn concentration.

The possibility that m-hydroxybenzaldehyde would also be hydroxylated in a similar manner by P. patulum mycel preparations also required investigation. Both Forrester and Gaucher [1,4] and Scott and Beadling [ 5 ] have implicated m-hydroxybenzaldehydc as bcing of significance in the biosynthesis of patulin, both by isotopic incorporation studies and by the presence of vary high levels of m-hydroxybenzaldehyde dehydro- genase activity. The interference of m-hydroxybenz- aldehyde in both m-crcsol and m-hydroxybenzyl alcohol 2-hydroxylase activities suggested that i t , too, might be a substrate for the possible single enzyme catalysing these reactions.

Incubation of 10000 x g supernatant preparations of the fungus with m-hydr~xy['~C]benzaldehyde in the presence of NADPH yielded large amounts of hydroxybenzyl alcohol due to the highly active de- hydrogenase, which is known to favour alcohol formation [4,5]. Another radioactive product was observed in smaller amounts on thin-layer chromatog- raphy of incubation extracts, but this did not corre- spond to the expected gentisaldehyde. Due to the difficulty of adequately separating m-hydroxybenz- aldehyde and gentisaldehyde by thin-layer chromatog- raphy, they were converted to their 2,4-dinitrophenyl- hydrazone derivatives prior to separation and testing for radioactivity. Negligible radioactivity could be detected in the area corresponding to gentisaldehyde 2,4-dinitrophenylhydrazone, even after longer term (30 min) incubations. Use of microsomal or washed microsomal preparations considerably reduced the conversion of m-hydroxybenzaldehyde to the alcohol, but incorporation into the other product was retained. Comparison of the mobility of this radioactive com- pound in four thin-layer chromatographic systems (Table 1) showed that the product was identical to m-hydroxybenzoic acid. This was confirmed by larger scale preparation and repeated recrystallisation as the rn-hydr~xy['~C]benzoic acid methyl ester, when a constant specific radioactivity was observed. An incorporation of 1.29 pmol m-hydroxybenzaldehyde x min-' x g dry mycel-' (75 mg microsomal protein) into m-hydroxybenzoic acid was observed in a prep- aration hydroxylating 0.89 pmol m-hydroxybenzyl alcohol x min-' x g dry mycel-I. Activity was entirely dependent upon the presence of NADPH.

DISCUSSION

The hydroxylation of m-hydroxybenzyl alcohol to form gentisyl alcohol has been shown to occur in particulate (microsomal) extracts from the fungus P. patulum. The activity was found to be typical of the

474 Microsomal Activities in Patulin Biosynthesis

Acetyl CoA + 3 Malonyl CoA

H CH3

6-Methylsalicylic acid ‘ 0 H -OH Y7-

rn-Hydroxybenzyl alcohol

llF COOH

@OHz &OH

11 dH m-Hydroxybenzoic acid m-Hydroxybenzaldehyde Gentisaldehyde Pat u L i n

big 4 Patulrri hrorjinthecrc in t e r m of r h ~ ~ enzjmrtully c ~ \ l u h ~ r ~ h r t / toni’er \ I U I I S of ihe known mrermedrure\ (A) Lynen and Tadd (1961). Scott er ol (1974) [16,17], (B) Light (1969), Vogel (1971) [18,3], (C,D) Murphyeiu l (1974) [ 6 ] , (La) this publication, (F) Forrester and Gaucher (1972), Scott and Beadling (1974) [4,5]; (H,I) Scott and Beadling (1974) [5]

mixed-function oxidase type, with a requirement for molecular oxygen and a rcduced cofactor, NADPH. Inhibition by carbon monoxide and its partial reversal by strong light suggested the involvement of a pigment like cytochrome P-450, as was described for the m-cresol hydroxylases [6 ] . This was supported by the implication of NADPH-cytochrome reductase partic- ipation in the hydroxylation mechanism, suggested by the inhibitory action ofcytochrome c and its reversal by cyanide ions. The action of other effectors was also similar for both rn-hydroxybenzyl alcohol and rn- cresol 2-hydroxylases, as was pointed out in the Results section, and suggested that the two activities might bc associated with one enzyme. Sucrose density gradient centrifugation failed to separate the two activities. If one enzyme is rcsponsible for both activ- ities is would thus have a poorer affinity for rn-cresol ( K , of 80 pM ; [6]) than for m-hydroxybenzyl alcohol (K , of 15 pM), reflecting the relative significance of the two activities in patulin biosynthesis, i.e. the 2,5-di- hydroxytolucne occurring in small amounts in P. patulum culture media is the result of m-hydroxy- benzyl alcohol hydroxylase action with m-cresol as the substrate.

No significant effect of other patulin biosynthetic metabolites on the activity of these microsomal rn-cresol and m-hydroxybenzyl alcohol hydroxylases was obscrved in vitro in the studies described. Further- more, the enzymes of the pathway are not induced by the addition of patulin precursors to cultures (the mycel appears to be completely permeable to most

of these intermediates) [4,5]. Bu’Lock et a/. [I41 have postulated that the regulation of patulin biosynthesis is effected by rapid turnover of the constituent enzymes, their synthesis being controlled by levels of common biosynthetic intermediates, e.g. acetyl-CoA, malonyl- CoA, which may build up at the transition to the Iodophase, and their subscquent utilisation for second- ary metabolism, possibly in conjunction with the appearance of the secondary metabolites which are their substrates.

The failure to detect an rn-hydroxybenzaldehyde 2-hydroxylase activity in cell-free preparations of P. patulum, under thc conditions employed in our laboratory, implies that patulin biosynthesis must proceed from m-hydroxybenzyl alcohol through gentis- yl alcohol, rather than m-hydroxybenzaldchyde to gentisaldehydc and patulin (Fig. 4). Alternatively the enzyme involved in m-hydroxybenzaldehyde-gentis- aldehyde conversion is very labile or different from the hydroxylases previously characterised in that it is soluble or requires soluble factors for its activity. Any activity masked in 10000 x g supernatant prep- arations by the high m-hydroxybenzaldehyde dehydro- genase levels would then be lost in washed microsomal preparations with much reduced dehydrogenasc inter- ference. An active NADPH-dependent conversion of m-hydroxybenzaldehyde to m-hydroxybenzoic acid was detectable however, which would account for the small pool of the latter that accumulates in culture media [I , 151. Scott et al. [2] reported that m-hydroxy- benzaldehyde was an efficient precursor of patulin,

C. Murphy and F. Lynen

although neither Scott and Beadling [ 5 ] , nor we could demonstrate direct conversion of the aldehyde to patulin. The possibility again exists that the enzyme involved is extremely unstable and, hitherto undetect- able in cell-free extracts.

The results of studies on the importance of gentisyl alcohol as a precursor of patulin, rather than as an end product, are controversial [I ,2]. However, Scott and Beadling [5] have demonstrated the presence of two reversible alcohol dehydrogenase activities favour- ing m-hydroxybenzyl alcohol and gentisyl alcohol formation from their respective aldehydes and were able to show that the rate of gentisaldehyde production from gentisyl alcohol in crude fungal extracts was of the same order of magnitude as the detectable gentisaldehyde to patulin conversion rate, i.e. gentisyl alcohol dehydrogenase is active enough to account for patulin synthesis if these measurements bear any relation to the rates in vivo. It is evident that a signifi- cant contribution to patulin biosynthesis could occur through the hydroxylation of m-hydroxybenzyl alco- hol to gentisyl alcohol, prior to reduction and oxidative rearrangement to form patulin, but the importance of rn-hydroxybenzaldehyde as a patulin precursor remains uncertain whilst only its conversion to m-hydroxy- benzyl alcohol and m-hydroxybenzoic acid are de- monstra ble.

G. Murphy was the recipient of a NATO science fellowship.

415

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G. Murphy and F. Lynen. Max-Planck-Institut fur Biochemie, D-8033 Martinsried, Am Klopferspitz, Federal Republic of Germany