Mycolic acid synthesis by Mycobacterium aurum cell-free extracts

~joc~irntca et Bio~~~sicu Art@, 1042 (1990)

315

315-323

Elsevier

BBALIP

53318

Mycolic acid synthesis by Mycobacterium Charlotte

Lacave, Marie-Antoinette

Centre de Recherches de Biochimie et G&hnque

Mycohc

LanCelIe and Gilbert

Cellulaires and UnrcersirP Paul Suhatw.

(Received

Key words:

aurum cell-free extracts

acid synthesis:

LanCelle Toulouse {France)

27 June 1989)

Cell-free synthesis:

(M~cobff~terium

uurum)

The first cell-free system capable of synthesizing whole mycolic acids: (R,CH(OH~CH(R~~COOH, with 60 to 90 carbon atoms) from fl-‘4C]acetate is described and preliminary investigations into some of its requirements and properties are reported. Biosynthetic activity for mycolic acids occurred in an insoluble fraction (40000 X g pellet) from disrupted cells of Mycobucterium aurum (ATCC 23366type strain); it produced mycolic acids, but a very small amount of non-hydroxylated fatty acids. The predominant product was unsaturated mycolie acid (type I), while oxo- (type IV) and dicarboxy- (type VI) mycolic acids were synthesized to a lesser extent. When II- “C]palmitic acid was used as a marker, no labelled mycolic acid was detected. The reaction required a divalent cation (Mg’+ or Mn”), KHCO, and 0,. Neither CoA, NADH, NADPH nor ATP were necessary, but CoA rather increased the synthesis of non-hydroxylated fatty acids. Glucose or trehalose were not required. Avidin inhibited the biosynthesis of the three types of mycolic acid indicating the presence of a biotin-requiring enzyme in the reaction sequence and therefore a earboxylation step, but citrate had no allosteric effect. Iodoacetamide inhibited the system. These first data are in favor of a complex multienzyme system.

Mycolic acids constitute the matrix of the lipidic part of Corynebacteria, Nocardiae and Mycobacteria cell walls, each genus producing characteristic types [l-3] which may be important to the pathogenicity and physiology of these micro-organisms [1,4,5]. Their biosynthesis is one of the targets of some antituberculous drugs [6-8] and understanding of the synthesis mechanism could offer new opportunities for interfering with mycobacterial metabolism in a specific manner. Obtaining isolated enzyme systems could provide a tool to decipher the action of some drugs and new possibilities to find novel chemotherapeutic agents against mycobacteria. Their general structure is R,CH(~H)~H(R~)COOH, in which two hydrocarbon chains R, and R, have variable lengths and R, (called the mero-chain) bears different functions (unsaturations and oxygenated functions). ‘True’ mycolic acids fall into the range of 60 to

Abbreviations: TLC. thin-layer chromatography; synthetase; PMSF, phenyimethylsulfonyl fluoride; tert-butylphenyl)-5-(4-biphenyl)-f-oxa-3,4-di~ole.

FAS. fatty acid butyl-PBD, 2-( p-

Correspondence: C. Lacave, Centre de Recherches de Biochimie Genetique Cellulaires and Universite Paul Sabatier. 118 route Narbonne. 31062 Toulouse Cedex, France. 0005-2760/90/$03.50

0 1990 Elsevier Science Publishers

et de

B.V. (Biomedical

90 carbon atoms and the structures of the three types present in M. aurum (ATCC 23366-type strain). namely unsaturated (I), oxo- (IV) and dicarboxy (VI) mycolic acids, have previously been described [3,9]. Many studies have been made to establish the structure [4,5,10] and biosynthetic pathway of mycolic acids [4,11] but many steps are still unknown [11,12]. Until now, most results have been obtained with intact bacteria [13,16]. Early studies with cell-free extracts of mycobacteria showed the biosynthesis of Cl6 to C32 fatty acids in M~~cobucterium smegmatis [17]. In 1984, Qureshi et al. [18] described the formation of longer fatty acids (40 to 56 carbon atoms). by a soluble cell-free extract of Mycobacterium tuberculosis H37 Ra. This system was able to synthesize labelled saturated and unsaturated straight-chain Cl6 to C56 fatty acids from [l-14C]malonate: the distribution of the products varied with the substrates used in the cell-free reaction. Some of the very long chain fatty acids had structures resembling that of the R, chain (mero) in mycolic acids, thus they might be their precursors, but the authors could not obtain complete mycolic acids with this cell-free extract. The general structure of mycolic acids suggested a biosynthetic route via a Claisen-like condensation reaction (‘mycolic condensation’) between two aliphatic chains followed by reduction of the 0x0 acyl formed [19]. Only the shortest mycolic acids (corynomycolic Division)

316 acids with 28 to 36 carbon atoms) were obtained from two aliphatic chains with cell-free extracts [20,22]. A soluble cell-free extract was first described [20] in Corynebacterium diphtheriae, able to condense two palmitic acid molecules into an 0x0 intermediate: 2-tetradecyl-3-octadecanoic acid, but without reduction into corynomycolic acid. Later, corynomycolic acid was synthesized with insoluble extracts from Bacterionema matruchotii [21,22]: condensation of two palmitic acid molecules into corynomycolic acid occurred with ‘a crude cell-wall fraction’ only in the presence of glucose (or of the 78000 x g supernatant). It seems likely that ‘mycolic condensation’ will be basically the same from Corynebacteria to Mycobacteria with R, in the general structure being palmitate in Corynebacteria and a characteristic mero-chain fatty acid in Mycobacteria. The present communication reports the first complete cell-free synthesis of three types of C60 to C90 mycolic acids from [l-‘4C]acetate as a precursor.

2-mercaptoethanol. A smooth suspension was prepared with a glass rod and then the cells were disrupted by two passages through a chilled French pressure cell (Aminco, Silver Spring, MD), at a pressure maintained above 200 MPa. Disrupted cells were first centrifuged for 15 min at 9000 X g and three fractions were obtained: (1) a white floating layer (probably equivalent to the ‘fluffy’ layer described by Shimakata et al. [21]), (2) a supernatant, and (3) a brown pellet. The supernatant was removed by decantation, the floating layer was carefully collected, smoothly homogenized with 20 ml of cold 0.05 M potassium phosphate buffer (pH 7.0), containing 5 mM 2-mercaptoethanol and centrifuged again at 1000 X g for 10 min to remove unbroken cells that could be trapped in the floating layer. Scheme I summarizes the principal steps to prepare the different fractions. Pl and P2 (Scheme I) were used as crude enzyme preparations within a day. Light and electron microscope checks were carried out after suspension of P2 in a suitable amount of water instead of phosphate buffer. For electron microscope examination, negative staining with 2% phosphotungstic acid (in water) was performed. Micrographs were made using a JEOL, JEM 1200 EX electron microscope. Enzyme ussuy. Mycolic acid synthesis was assayed as follows: The standard reaction medium (1.5 ml) was adapted from the assay used with the C. diphtheriue cell-free system [20]. It contained 67 pmol potassium phosphate buffer (pH 6.0), 5 pmol MgC12, 10 pmol KHCO, and 200 nmol of [I-i4C]acetic acid (sodium salt, spec. act. 2 GBq/mM). The reaction was started by addition of enzyme extract (0.8 to 1.2 mg of protein were used per assay, the protein was determined by Lowry’s method [23] using bovine serum albumin as standard) and incubated for 60 min at 37°C under agitation (shaker incubator, model G24. New Brunswick) at 150 oscillations per min. The reaction was terminated by the addition of 1.5 ml of 20% KOH in

Materials and Methods Chemicals. ATP (disodium salt) NADH, NADPH, DNase were obtained from Boehringer. D-Biotin, avidin, CoA, PMSF and bovine serum albumin were purchased from Sigma. EDTA (Titriplex III) was from Merck, iodoacetamid from Serva, 2-mercaptoethanol from Calbiochem and butyl-PBD from Koch-Light. Prepuration of extracts. M. aurum (ATCC 23366) cells were grown on a medium containing glucose as previously described [16] and were harvested in the mid-log phase. After centrifugation at 4°C the cells were washed once in cold 0.01 M potassium phosphate buffer [pH 7.01. All the following procedures were carefully carried out at 4” C. About 5 g (drained weight) of packed cells were suspended in 25 ml of cold 0.05 M potassium phosphate buffer (pH 7.0) containing 3 mM

Cell suspension (after French (about 25 ml)

pressure

cell dlsruptlon)

Pellet (unbroken cells) dvscarded

II

Pellet P2

Scheme

supernotont

Pellet (unbroken cells) d,scorded

Supernotant

Pellet (unbroken cells) d Iscorded

Pellet

Supernatont

!A

3-C

I. Preparation

SL

of the cell-free

extract

supernotont s3

317

Fig. 1. Electron

micrograph

of a cell-free

fraction of M. autwn prepared wth French pressure cell. The specimens JEM 1200 EX electron microscope. The bar shows 100 nm length.

methanol/ benzene (8 : 2, v/v). A glass marble was placed on each tube prior to overnight saponification at 75-80 o C. Extraction and separation of the lipids. After cooling and removal of methanol under nitrogen, the content of each tube was acidified by 20% H,SO, and extracted three times with 2 ml of diethyl ether. The extract was washed three times with water, dried and aliquots (generally 1/50th of the total vol.) were counted in butylPBD using a Intertechnique scintillation spectrometer SL30. Lipids were analyzed by thin-layer chromatography, on Silica gel G60 plates (Merck). All runs were performed in lined tanks with the following solvents: A, light petroleum/diethyl ether (7: 3, v/v); and B, dichloromethane. Two TLC separations were systematically performed: the first before methylation of the whole extract (solvent A) to visualize non-polar compounds such as methyl-carbinols (2-octadecanol liberated from wax ester by vigorous hydrolysis [16]) and eventually long-chain ketones; the second after methylation by diazomethane (solvent B) to identify the three types (I, IV, VI) of mycolic esters known in M. aurum [9] by co-chromatography with known standards extracted from whole cells. Some unidentified polar lipids were always present just above the origin of the chromatogram that could be similar to hydroxylipids quoted by Qureshi et al. [18]. Radioactivity was located and counted on plates using an automatic TLC linear analyzer (Berthold LB 2832). Then, the lipids were visualized by spraying Rhodamine B, then 20% H,SO, followed by charring. Definition of the activity of the system. It was defined as the quantity of [l-‘4C]acetate incorporated per mg of

were examined

with a JELL

protein for 60 min and 100% activity was adopted for the value obtained in standard conditions described in the assay method. It corresponds to the incorporation of 30 nmol (f 2) of acetate per mg protein for 60 min and was determined by direct counting of the extracts (1/50th aliquot). The other values were expressed as percentages of the standard values. Enzyme stability. It was noted in preparation of cellfree extracts that they had to be used within 1 day. Examination of storage conditions had shown that storage of preparations led to considerable loss of activity. Table I summarizes the principal results. It must be mentioned that Pl (see Scheme I) kept a better activity than P2, so purification seemed to increase the instability of the enzyme system. Bloch et al. underlined that the multienzyme complex FAS I [17] was more stable when stored in a high ionic strength buffer. Our attempts at this did not give any difference, either for Pl TABLE Influence

P2

Pl

I of storage condrrrons

Conditions

% Activity

Just after centrifugation 2hat20”C 15hatO”C 1 weekat -20°C 1 monthat -70°C 1 month at -70°C+2

mM PMSF

100 75 15 3 10 10

Just after preparation 1 month at -70°C+2

mM PMSF

100 60

’ 100% activity corresponds the standard conditions.

to the immediate

a

use of the extracts

in

318 or P2 and until now, no stabilizing factors have been found. Miscellaneous. Assay controls with intact bacteria: 400 nmol [l-‘4C]acetate (spec. act. about 2 GBq/mmol, CEA, Saclay, France) were added to 100 ml culture of M. aurum in mid-log phase of growth and aliquots of 10 ml were treated as previously described [16]. Analyses of the extracts were performed by direct counting and TLC as mentioned above for assays with cell-free extracts. Results and Discussion

Microscopic and ultramicroscopic checkings These showed that most of the whole cells had been removed. Most cells were disrupted as can be seen in Fig. 1, often into ‘half-cells’. The appearance after disruption of M. aurum in a French-press is quite comparable to that described for cells of Bacterionema matruchotii disrupted by the same procedure [22]. Incorporation of acetate into mycolic acids by an insoluble cell-free extract [l-‘4C]Acetate was not incorporated into lipids by the supernatant fractions (Sl, S2, S3 in Scheme I) whatever compounds were added (ATP, NADH, NADPH, CoA, glucose) (Fig. 2A). In M. tuberculosis H37 Ra, the same observation was reported [18]: acetate could not be used by the soluble enzyme system. With the insoluble preparations (Pl or P2, Scheme I) a quite notable incorporation of acetate into lipids was observed (by direct counting and by radio-thin-layer chromatography) when the cells were harvested in midlog phase growth. In stationary phase, the activity of the cell-free extracts was much lower as noted previously by Bloch et al. [17] with the fatty acid synthetase system of M. smegmatis. A first run on thin-layer plates with the whole extract before esterification (solvent A) showed that almost all the label was in the fatty acids (Fig. 2B, peaks 1 and 2) and only a small amount was found in the ketones (Fig. 2B, peaks 4 and 5) while 2-octadecanol (spot 3 in fig. 2B) was not labelled. This alcohol resulted from the saponification of mycolic wax constituted by dicarboxymycolic acid esterified on its o-carboxyl group by this octadecanol [24]. Analysis of esters showed an active synthesis of mycolic acids (Fig. 2D, peaks 3-6) whereas only a minor labelling of non-hydroxylated fatty acids (peak 7, Fig. 2D) could be detected. The same profiles were obtained with Pl and P2 except a greater labelling in dicarboxymycolic acid with Pl and a more intense peak of non-hydroxylated fatty acids. The results being more reproducible with P2 (obtained after a 40000 x g centrifugation which removed some soluble compounds, see Scheme I), the following tables only concern results obtained with this fraction except when otherwise stated.

Fig. 2. Comparison of in wtro and in viva profiles on thin-layer plates: A. in supernatant Sl after esterification; B, in vitro, C. in viva (both before methylation solvent A). 1, mycolic acids + unidentified polar acids: 2, non-hydroxylated fatty acids; 3, 2-octadecanol; 4 and 5, long-chain ketones. D, in vitro, E, in viva (both after methylation solvent B). 1 and 2, unidentified polar esters: 3. 2-octadecanol; 4, dicarboxymycolic ester: 5. oxomycohc ester; 6. unsaturated mycolic ester; 7, non-hydroxylated short fatty esters (up to C24); X, non-hydroxylated mero-esters ( + ketone 4 of Fig. 28); 9. ketone 5 in profiles B and C. With TLC radioscanning of the radioactive products, spots were visualized (and identified after co-run with known standards) wuth Rhodamine B spray followed by 20% HaSO, and charring. Radioactivities were monitored with a radioactivity scanner (Berthold LB 2832).

As mentioned earlier, some unbroken cells might remain, but the following results brought evidence that activity could not be attributed to whole cells.

319 TABLE

II

[‘4C]Acetnte-labelling

of mycolates and other saponification

products

in whole cells and in cell-free extracts

Results are expressed as percentage of radioactivities detected by radioscanner on thin-layer plates. On the left part of the table. 100% of radioactivity represents the sum of labelled compounds detected on the plate and the ratio mycolic acids/non-hydroxylated fatty acids is the ratio of the percentages of these acids, respectively. On the right part of the table. 100% represents the sum of the radioactivities of the three mycolic acids (unsaturated + oxo- + dicarboxy-mycolic acids = 100%). The figures were obtained with ten assays in vitro and four in viva. % of total radioactivity neutral compounds

’ polar’ esters h

mycolates ’

non-hydroxylated fatty acids d

15 5

65-70 35

5-10 50

a

Ratio of mycolic acids to non-hydroxylated fatty acids

Mycolate unsaturated mycolates

oxomycolates

dicarboxymycolates

7-10 0.8

55-65 25-30

25-35 60-65

2-10 5-10

relative labelling

Ratio of unsaturated mycolates to oxomycolates

(%)

Cell-free extract Whole cells

10 10

2.2-2.6 0.5-0.8

a In vitro labelled neutral compounds: ‘ketones’ (peaks 4 and 5 Fig. 2B). In vivo labelled neutral compounds: 2-octadecanol (peak 3. Fig. 2C). ’ Unidentified polar esters (peaks 1 and 2 in Figs. 2D and 2E). ’ Sum of the three mycolates (diunsaturated (peak 6, Figs. 2D and E) + oxo-(peak 5, Figs. 2D and E) + dicarboxy-mycolates (peak 4, Figs. 2D and E)). d Non-hydroxylated fatty acids (up to 24 carbon atoms): Correction was made for spot 7 (Fig. 2D) which contains

TABLE Effects

peak 7 in Figs. 2D and E and unsaturated neutral ketones (peak 4, Fig. 2B).

meromycolic

acids

(peak

8, fig. 2D).

III of addition or deletion of rnorganrc

compounds

The standard system contained 67 pmol potassium phosphate buffer, 10 pmol KHCO, 5 ymol MgCl,, 200 nmol [l-‘4C]acetate and 0.8 to 1.2 mg protein (P2) in a final volume of 1.5 ml. Relative percentages always represent averages of four experiments and variations represent about 10% on each value because of the difficulties in perfect homogenisation of cell-free preparations. Percentage activity also represents the average of four experiments. Values can be considered as significantly different if they differ by 15 to 20%.

Potassium phosphate 0.05 M (pH 6.0) 0.2 M (pH 6.0) 0.05 (pH 7.5)

% of total radioactivity

’

dicarboxymycolic acids

oxomycolic acids

unsaturated mycolic acids acids

9 11 15

19 21 20

40 40 32

nonhydroxylated fatty

Ratio of unsaturated mycolic acids to oxomycolic acids h

Ratio of mycolic acids to non-hydroxylated fatty acids h

% activity

buffer: 8 8 8.5

2.1 1.9 1.6

8.5 9 8.1

100 120 70

no KHCO,

6.3

17

45

13

2.6

5.3

80

no Mg2+

6

18

44

13

2.4

5.2

75

no Mg’+ and no HKCO, Mn*+ instead of Mg*+

_

_

_

_

5.6

8

44

4.2

_ 2.2

5

18

28

9

1.6

5.6

95

8

27

34

14

1.3

4.9

30

added

EDTA 0.01 M

no 0,

’

17

40 75

Standard

conditions

More labelling in dicarboxymycolic acids Less labelling in mycolic acids Less labelling in mycolic acids d

Less labelling in oxygenated mycolic acids Less labelling in mycolic acids ’ Strong inhibition of mycolic acid synthesis

a Results on the left part of the table are expressed as relative percentages of radioactivity measured on thin-layer plates; the difference with 100% results from polar compounds (1 and 2 in Fig. 2D) and neutral ketones (4 and 5 in Fig. 2B). ’ Ratio of radioactivity percentages. ’ 100% activity is defined for the standard conditions as described in the text after counting of the total radioactivity per mg protein for 60 min. ’ The relative percentages were not registered. ’ EDTA 0.01 M was added to the buffer before disruption of the cells.

320 sions: the ratio of relative percentages of radioactivities between unsaturated and 0x0 mycolic acids is characteristic: lower than 1 with intact bacteria, greater than 2 in cell-free extracts, as it appears in the following Tables. In the same way, the ratio of relative percentages of radioactivities between the sum of mycolic acids and non-hydroxylated fatty acids was below 1 in intact bacteria, but close to 10 in cell-free extracts. This last ratio gives an excellent indication of the cell-free extract activity. Time course of futty acid .synthesis As shown in Fig. 3 the synthesis of mycolic acids was dependent on the incubation time. The linear portion of the curve was between 30 and 120 min. It must be underlined that the radioactivity in the dicarboxymycolic acids was high after 120 min (not shown); this is in good agreement with the mechanism proposed for its formation [16,25], i.e., oxidation of the 0x0 mycolic acids by a Baeyer Villiger process.

hours Fig. 3. Time dependence of incorporation of [1-‘4C]acetate into mycolic acids by the cell-free extract P2 (see Scheme I). Assays were as described under ‘Materials and Methods’ except that the time was changed as indicated in the figure.

Fig. 2 and Table II show that the activity in the extracts is distinct from the activity in intact cells: non-hydroxylated fatty acids (Fig. 2E, peak 7) were the major acetate-labelled compounds and the oxo-compound (peak 5) was the prominent labelled mycolic acid with intact bacteria, while the unsaturated mycolic acid (peak 6) was the major one in the cell-free extracts. Table II gives a quantitative basis to the above conclu-

TABLE

Effect of uddition or deletion of different inorganic fuctors (T&e III) Conditions for a good enzyme assay were established and an activity for the enzyme complex defined. No added factor was needed either with Pl or with P2. A pH of 6.0 gave a better total incorporation of acetate with maximum mycolic acid labelling, though dicarboxymycolic acid radioactivity was lower than at pH 7.5,

IV

.q$ecr of SOnW cofuctors For legends and footnotes

see Table III

% of total radioactivitv

Ratio of mycolic acids to non-hydroxylated fatty acids h

% activity

unsaturated mycolic acids

9 9

19 19

40 40

8 9

2.1 2.1

8.5 7.8

100 110

CoA (1 mmol)

11.5

19

36.5

6.3

1.9

10.5

175

ATP + CoA Biotin (20 pg)

13 11.5

18.5 16.5

37 38

1.3 4.2

2 2.3

9 15

150 120

9 12.8

16 19

41 36.6

10 5.3

2.6 1.95

6.6 12.4

15 50

110

Standard conditions ATP (5 pmol)

Biotin + ATP Avidin (1 unit)

f

non-hydroxylated fatty acids

Ratio of unsaturated mycolic acids to oxomycolic acids ’

oxomycolic acids

dicarboxymycolic acids

Avidin (2.5 units) ‘J _

_

_

15

(4) Avidin + biotin s

11.3

18.2

38

6

2

11.2

Citrate (10 pmol)

10

15

45

5

3

14

&r Cell-free extract pre-incubated with avidin 10 min before 8 Avidin and biotin were pre-incubated 10 min at 37 o C.

addition

of the other components.

80

’

More labelling in polar esters More labelling in mycolic acids More labelling in mycolic acids

Strong inhibition of all syntheses

321

but only maximum total mycolic acid synthesis was studied in this work whatever the distribution between the three types of mycolic acids was. MnCl, may replace MgCl, but it strongly inhibited labelling of oxygenated mycolic acids so MgCl, was routinely added. Its presence as well as this of KHCO,, favoured whereas their double deletion mycolate synthesis, strongly decreased it. EDTA added to the buffer before cell disruption slightly inhibited mycolate synthesis, so it was not used in standard conditions. Lowering the amount of 0, by bubbling N, through the assay tube abruptly inhibited mycolate synthesis. Influence

of addition of some cofactors (Table IV)

As previously mentioned, no cofactor was strictly required for activity, but addition of some of them modified the incorporation of acetate into the mycolic acids (Table IV): CoA gave the best labelling (175% of standard activity). Biotin enhanced mycolic acid synthesis to a lesser extent, but it is worth noting the strong inhibition by avidin (50 to 85% according to the relative concentrations), the ratio of radioactivities of mycolic acid to non-hydroxylated fatty acids is 5.3, whereas preincubation of avidin with biotin abolished its inhibitory effect. A control with intact bacteria was

TABLE Effects

avidin had no effect on in vivo also performed: syntheses. This result confirmed the existence of a cellfree system. The inhibitory effect of avidin on cell-free extracts strongly indicated the presence of a biotin-containing enzyme in the reaction sequence, which may be assumed to be a carboxylase [26]. A carboxylation step seems to be essential for the complete synthesis of mycolic acids. As citrate is known as an allosteric effector of carboxylation enzymes [27], its addition was tested, but no effect was detected, except for a slight inhibition of the total activity (80%). It was noticeable that ATP was not required, addition of this energy factor did not improve synthesis either because the system contained a sufficient amount of ATP, or already had ATP.-producing systems to accomplish mycolic acid synthesis. Such a phenomenon has already been described in mycobacteria for the multienzyme FAS I of M. smegmatis [17]. It is also possible that another factor was used since in some cases, pyrophosphate [28] or polyphosphates [29] can serve as an energy donor. Similarly, addition of NADH, or NADPH did not improve the system. Effect of some substrates (Table

Addition of glucose described as activating

V)

to enzyme assays was often mycolic condensation [20,22].

V of addition of mme substrates

For legends

and footnotes

see Table III.

% of total radioactivity

’

dicarboxymycolic acids

oxomycolic acids

unsaturated mycolic acids

9

non-hydroxylated fatty acids

8

Ratio of unsaturated mycolic acids to oxomycolic acids ’

Ratio of mycolic acids to non-hydroxylated fatty acids h

% activity

’

No addition Glucose (10 pmol)

12

19 19.5

40 38

I

2.1 1.95

8.5 9.9

100 60

Glucose (10 pmol) + ATP (5 n mol)

12

18.5

38.5

8

1.95

8.6

50

Less labelling in mycolic acids

Trehalose (10 wmol)

14

22

37

7.5

1.7

9.7

95

More labelling in dicarboxymycolic acid

1.4 1.4

16.7 17

34 34

9.2 8

2 2

6.4 6.7

105 95 )

9.4

16.5

34.2

20

2.1

3.0

180

More labelling in non-hydroxylated fatty acids with Pl

8

15

38

11.4

2.3

5.4

120

Weak activity

0

0

0

+

_

_

Malonate

(2.5 pmol) (12.5 gmol)

CoA + malonate (2.5 umol)

No activation

+ Pl

CoA + malonate + P2 (2.5 pmol) [l-‘4C]Palmitic acid (50 nmol) ’

r [l-‘4C]Palmitic

Standard conditions Less labelling in mycolic acids

acid was used instead

of [l-‘4C]acetate

after purification

by TLC

Presence esters

on P2

of polar

322 TABLE

VI

Effec,/ o/ clddrtron of some inhihlrou For legends and footnotes

see Table III

% of total ratioactivity

No addition s1t Pl Sl + P2 Boiled Sl + P2 Bolled Sl + Pl M ‘+P2 Iodoacetamide + P2 ’ M represents

a

dlcarhoxymycolic acids

OXO-

mycolic acids

unsaturated mycolic acids

non hydroxylated fatty acids

9 10 8 11.5 11 10.5 0

19 29 26.5 15 17 14.5 0

40 29 33 30 32 33 0

8 14.5 11 14 13 11.2 traces

pellet after centrifugation

of Sl at 40000~

g (see Scheme

Table V shows that glucose was rather inhibitory with the cell-free extract of M. aurum. Trehalose had no effect nor did malonate at different concentrations. However with Pl, malonate and CoA acted together as a strong activator of incorporation of acetate into nonhydroxylated fatty acids. but not into mycolic acids; it is possible that some soluble FAS was present in PI, while after 40000 x g centrifugation this amount was much lower. Qureshi et al. [18] observed, in the presence of malonate (and malonyl-CoA) the formation of long non-hydroxylated acids (C40 to C56). and earlier Bloch et al. [17] had shown that modifications of the relative concentrations of acetate and malonate brought severe changes in the pattern of synthesised fatty acids. Indeed an increased labelling of non-hydroxylated fatty acids was observed in the presence of malonate (Table V). Palmitate was not incorporated by the cell-free extract of M. uurum, either into mycolic acids or into non-hydroxylated fatty acids, yet it was shown with intact bacteria that palmitate was incorporated into both the side chain (R,) and in the mero-chain (R,) of unsaturated mycolic acids [ 131.

Stud of some inhibitors Table VI summarizes the effect of some factors on the system. It must be noticed that addition of Sl (supernatant, Scheme I) strongly inhibited both Pl and P2 activities. Activity might be completely lost with the right relative concentrations of P and S. The inhibitory factor seemed to be thermostable since boiled Sl was still efficient. Inhibition was stronger for mycolic acid synthesis as was shown by the values of the ratio of radioactivities of total mycolic acid to non-hydroxylated fatty acid. After 40000 x g centrifugation of Sl, the pellet M (Scheme I) had kept all the inhibitory proper-

Ratio of unsaturated mycolic acids to oxomycolic acids h

Ratlo of mycolic acids to non hydroxylated fatty acids h

2.1

8.5 4.7 6.1 4 4.6 3.4

1.2 2 1.9 2.3

% activity

’

(3)

100 25 20 15 1 20 i ‘51 15

Standard

Strong

conditions

Inhibitton

I).

ties (Table VI) this pellet was probably a membrane fraction. No incorporation of acetate was detected in the presence of iodoacetamide, a sulfhydryl reagent.

Conclusion

The first cell-free system able to synthesize the whole ‘true’ mycolic acids from [l-‘4C]acetate as labelled precursor is described. As underlined at the beginning, the recorded mycolate synthetase activity could not be attributed to whole cells even if some were present in the extract (Fig. I). The main evidence is: the great differences between in vitro and in vivo TLC profiles of labelled molecules (Fig. 2) and the values of the two radioactivity ratios (unsaturated mycolic acid/oxomycolic acid, and total mycolic acid/non-hydroxylated fatty acids); the rather fast loss of activity; the absence of labelled 2-octadecanol in the cell-free extracts while this alcohol was strongly labelled in intact bacteria [16]; differences of distribution of acetate-labelled lipidic compounds in the whole cells and in the cell-free system (Table II), the latter being more close to the composition of the cell wall fraction [16]; avidin inhibition; Sl and M inhibitions and the absence of incorporation of palmitic acid into mycolic acids in the cell-free extracts. Cofactors that increased incorporation were not very effective for mycolate synthesis but rather for other components: either non-hydroxylated fatty acids or unidentified polar compounds. Biosynthesis of mycolic acids must include many steps: elongation and condensation reactions with several intermediate sequential reactions such as transacylations, carboxylations, reductions, dehydrations, de-

323 saturations performed

etc, so it is likely that complete synthesis by a complex multi-enzyme system [30].

is

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