/ . Biochem., 78, 381-390 (1975)

Pyridine-2, 6-dicarboxylic Acid (Dipicolinic Acid) Formation in Bacillus subtilis II. Non-enzymatic and Enzymatic Formations of Dipicolinic Acid from a, e-Diketopimelic Acid and Ammonia Kinuko KIMURA and Taiji SASAKAWA1 Laboratory of Biochemistry, College of Science, St. Paul's University, Nishi-Ikebukuro, Toshima-ku, Tokyo 171 Received for publication, October 23, 1974

Non-enzymatic formation of dipicolinic acid (DPA) from diketopimelic acid and ammonia was clearly demonstrated using a new method for DPA analysis. The reaction rates of DPA formation were almost the same under aerobic and anaerobic conditions. Nearly equimolecular quantities of DPA and tetrahydrodipicolinic acid were detected in spontaneous reaction mixture. The spontaneous reaction seemed to be due to dismutation of dihydrodipicolinic acid, resulting in DPA and tetrahydrodipicolinic acid. The apparent optimum pH of the spontaneous reaction was 8.2 and the maximal rate of DPA formation was observed with a 1: 4 molar ratio of diketopimelic acid to ammonia. The rate of the spontaneous reaction was stimulated by ferrous sulfate, FMN, and riboflavin. Dihydrodipicolinate reductase catalyzes the reduction of dihydrodipicolinate, prepared from pyruvate and aspartic /3-semialdehyde, with NADPH as reductant. The reductase was isolated from Bacillus subtilis, and found to stimulate DPA formation from diketopimelic acid and ammonia. The enzymatic DPA formation was absolutely dependent on oxygen, and optimum pH was 6.4. The catalytic action of the enzyme was similar to that of the oxidase. Possible mechanisms of DPA formation from diketopimelic acid and ammonia are proposed.

Previous studies from this laboratory have shown that at least two of the chemical condensation products of pyruvate-aspartic semialdehyde are active precursors of dipicolinic acid (DPA). These precursors, named dihydrodipicolinic acids, are converted to DPA spontaneously at pH 6.1. It seems likely that one 1

Deceased on October 6, 1973.

Vol. 78, No. 2, 1975

of these compounds is identical with the enzymatic condensation product ( / ) . On the other hand, a, £-diketopimelic acid has been suggested to be a possible precursor of DPA by Powell and Strange (2). They found that this keto acid is spontaneously converted to DPA in the presence of ammonia. Crude bacterial extracts had a catalytic effect on this reaction, which they ascribed to the presence 381

382

K. KIMURA and T. SASAKAWA \

Hz

H2 NH3

H

" S CO2H

HOzC

O2H

\ a,e-Diketopimelic acid

1,4-Dihydrodipicolinic acid

Oipicolinic acid

Scheme 1. Synthesis of dipicolinic acid from a, £-diketopimelic acid and ammonia (Powell & Strange, 1959).

of heat-stable factors. They also postulated that 1,4-dihydrodipicolinic acid, the immediate precursor of DPA, could be oxidized to DPA with oxygen (Scheme 1). Doi and Halvorson demonstrated that soluble NADH oxidase of spores in Bacillus cereus T stimulated oxygen uptake by a reaction mixture containing DPA (3). The formation of diketopimelic acid in spore-forming bacilli has not yet been demonstrated, and the physiological role of the keto acid as a precursor of DPA has not been considered. To obtain information on the nature of dihydrodipicolinic acid, DPA formation from diketopimelic acid was re-examined. This paper describes studies on the spontaneous conversion of diketopimelic acid and ammonia to DPA, and on the catalytic effect of dihydrodipicolinic acid reductase of Bacillus subtilis on the formation of DPA.

Assay Methods — The standard reaction mixture for DPA formation contained in a final volume of 1 ml: 5 /amoles of diketopimelic acid, 10 //moles of ammonium sulfate, and 50 //moles of phosphate buffer, pH 6.4 with or without the enzyme. The mixture was incubated at 37° for 60 min, unless otherwise indicated. DPA was estimated by the specific UV absorption of the Ca-DPA complex ( / ) . The incubation mixture was immediately diluted with cold 0.02 M CaCl2 solution and the amount of DPA was determined by the difference in the absorbancies at 277.5 and 274 nm, to avoid the influence of UV absorption of possible contaminants in this wavelength range. In some experiments DPA was estimated directly from the increase in absorbancy at 270 nm. Reaction products other than DPA formed from diketopimelic acid and ammonia, were analyzed by the acid ninhydrin reaction (6) and by formation of complexes with 0-aminobenzaldehyde (7). The acid ninMATERIALS AND METHODS hydrin reaction was carried out as follows. A sample of 0.1 ml of reaction mixture was Materials—Crystalline diketopimelic acid evaporated to dryness in vacuo at 60°. Then (mp 128—129°) was synthesized from diethyl 3.95 ml of glacial acetic acid, 0.2 ml of ninoxaloacetate by the method of Cope and hydrin solution (18.6 mg/ml of glacial acetic Fournier (4). Catalase [EC 1.11.1.6], prot+ + acid), and 0.05 ml of water were added. The amine sulfate, NAD , NADH, NADP , and mixture (in a glass-stoppered tube) was heated NADPH were products of Sigma Chemical Co. in boiling water for 8 min and then cooled in The other chemicals used were commercial, ice and the absorbance at 420 nm was measreagent grade compounds. ured. The reaction with o-aminobenzaldehyde Preparation of Dihydrodipicolinic Acid was carried out as follows. A sample of 1.0 Reductase of Bacillus subtilis—Dihydrodipicoliml of reaction mixture was mixed with 1.0 ml nic acid reductase was purified from cell free of 1 N HC1 and 1.0 ml of o-aminobenzaldehyde extracts of sporulating cells of Bacillus subtilis reagent (2 mg/ml of water) and incubated at PCI 219. The purification and properties of 37° for 90 min. Then the absorption at 460 the enzyme were reported previously (5). The nm was measured. All spectrophotometric reductase contained two moles of FMN per measurements were made with a Hitachi, mole. / . Biochem.

DIPICOLINIC ACID FORMATION FROM DKP AND AMMONIA

383

model 323 or 181, spectrophotometer. DPA formation from diketopimelate begins immediately after addition of ammonia, so anaerobic reactions were carried out using Thunbergtype quartz cells. RESULTS Non-enzymatic Formation of Dipicolinic Acid from Diketopimelic Acid and Ammonia— As reported in the literature (2, 3), a, £-diketopimelic acid is readily converted to DPA in the presence of ammonia. In this reaction, 1,4-dihydrodipicolinic acid is the most possible intermediate, but it is unstable and not characterized. In Fig. 1, the increase in UV-absorption at 335 nm may be due to the formation of this intermediate, which shows an analogous spectrum to those of the reduced forms of pyridinenucleotides. Diketopimelic acid itself has no measurable absorption in the UV region longer than 240 nm, but its sodium salt has a weak absorption at about 320 nm. This absorption may be characterized by the isomerization of the keto acid to the enol form. The increased UV absorption at 270 nm during the reaction (Fig. 1) was characteristic of the formation of DPA. The reaction mixture was applied to a Sephadex G-10 column and eluted with 0.02 M CaCl2, as previously reported ( 1 ) . The mate-

O.8-

240

260

280

300

320

o z < m a.

o

to CD

B

0.3



\

/ I n No.e

0.2



0.1

2

4

6

6

10 12

FRACTION NUMBER



250

270

290

310

WAVE LENGTH (nm)

Fig. 2. Gel nitration of the reaction products on Sephadex G-10 column. The reaction mixture contained, in a final volume of 1.0 ml, 100 //moles of Tris-HCl buffer, pH 8.2, 3 //moles of diketopimelate, and 10 //moles of ammonium sulfate. Incubation was carried out at 37° for 60 min. 0.5 ml of the incubation mixture was subjected to gel-filtration on a Sephadex G-10 column and the absorption of each fraction at 270 nm was measured. A: gel filtration profile; B: UV-spectrum of fractions No. 3 and 8. Vol. 78, No. 2, 1975

360

380

Fig. 1. Change in the UV-absorption after incubation of a, £-diketopimelate with ammonium sulfate. The reaction mixture (8.0 ml) containing 1.6 mM diketopimelate (neutralized with NaOH) and 2.6 mM ammonium sulfate was incubated at 37°. Spectra were recorded at the times indicated. Keto acid was measured with 2,4-dinitrophenylhydrazine (P).

I 0.3 •

340

W A V E LENGTH ( n m )

384

K. KIMURA and T. SASAKAWA

rial with UV-absorption was eluted at a KA of about 1 (Fig. 2A). The UV-absorption spectrum of the main peak was similar to that of the Ca-DPA complex, as shown in Fig. 2B. Optimum pH and Dependence on the Amount of Ammonia—As shown in Fig. 3 the pH optimum of the spontaneous reaction of net synthesis of DPA was found to be at about pH 8.2. It can be assumed that the DPA formation from a, e-diketopimelic acid with ammonia proceeds in a two-step reaction, according to Scheme 1. The pH dependence of the reaction rate of each step could not be estimated, but the maximal increase of A3t0 at about pH 7 (Fig. 3) was explained by assuming that the ratio of &1/&2 was maximum at this pH (&! and k2 were the rate constants of the 1,4-dihydrodipicolinic acid formation and DPA formation, respectively). Figure 4 demonstrates that with a fixed amount of diketopimelic acid the reaction rate was dependent upon the amount of ammonia. DPA formation was optimal with a 1 : 4 molar ratio of diketopimelic acid to ammonia at pH

0.25

Stimulatory Effects of Ferrous Sulfate and FMN—The stimulatory effect of heated cellfree bacterial extracts on DPA formation from diketopimelic acid and ammonia had been ascribed to the effect of some heat-stable factors in the extracts (2). Stimulation by FMN has also been reported (3). As shown in Figs. 5 and 6 respectively, ferrous sulfate and FMN stimulated the rate of spontaneous DPA formation. The effects of these two compounds were additive (Table 1, Reaction (b)). FMN might cause stimulation by acting as a catalyst in the oxidation of a precursor of DPA. Free riboflavin was also stimulatory. Previous work reported from this laboratory showed that mercuric ion has a stimulatory effect on the non-enzymatic DPA formation from the condensation products of pyruvate with aspartic jS-semialdehyde. However, it caused no stimulation under the present conditions (1). Analysis of Reaction Products Other Than DPA Formed on Incubation of Diketopimelic Acid with Ammonia—The fact that DPA is formed spontaneously during strictly anaerobic incubation of diketopimelic acid with ammonia

2.0

pH8 2 0.20

- 0.04

o is •

- 0.03

0.10

- 0.02

5

10

15

(NH«)sS04

Fig. 3. Optimum pH of DPA formation. The reaction mixture contained, in a final volume of 1.0 ml, 100 //moles of phosphate buffer, 5 //moles of diketopimelate, and 10 //moles of ammonium sulfate. It was incubated at 37° for 60 min. Samples of 0.1 ml of incubation mixtures were diluted to 3 ml with H2O for measurement of absorption at 270 (O) and 340nm ( • ) .

20

29

(.u moles)

Fig. 4. Effect of ammonium sulfate concentration on DPA formation. The reaction mixture contained, in a final volume of 1.0 ml, 100 //moles of Tris-HCl buffer, 5 //moles of diketopimelate and the indicated amount of ammonium sulfate. Mixtures were incubated at 37° for 60 min. The amount of DPA was calculated from the absorption using the formula DPA/ml). ]. Biochem.

DIPICOLINIC ACID FORMATION FROM DKP AND AMMONIA

385

TABLE I. Detection of Tetrahydrodipicolinate. The reaction mixture was as for Fig. 11. After incubation for 60 min at 37°, 0.1 ml of the mixture was used for the acid ninhydrin reaction and the rest (0.9 ml) for reaction with o-aminobenzaldehyde. The procedure was as described in the " MATERIALS AND METHODS."

Reaction mixture 100

FMN (n moles)

Fig. 5. Effect of FMN on DPA formation. The incubation mixture contained, in 1.0 ml, 70//moles of Tris-HCl buffer, pH 7.8, 5//moles of diketopimelate, 10 //moles of ammonium sulfate and the indicated amount of FMN. Mixtures were incubated at 37° for 60 min and then 0.5 ml of each mixture was subjected to gel filtration. The amount of DPA was calculated from the absorption.

FeS04 (.u moles)

Fig. 6. Effect of FeSO4 on DPA formation. The incubation mixture and conditions were as for Fig. 5, except that the indicated amount of FeSO4 was used instead of FMN.

strongly suggests the occurrence of coupled oxidation and reduction of some intermediate. As proposed in the literature (.?), the most likely dismutation is that of dihydrodipicolinic acid and this would result in the formation of DPA and perhaps tetrahydrodipicolinic acid. To test this the reaction mixture was subjected to the acidic ninhydrin test and color reaction with o - aminobenzaldehyde, as described under "METHODS." The results in Table II show that another product besides DPA was formed, which reacted in both color reactions. The products were partially separated by gel filtration of the reaction mixture on Sephadex G-10 (Fig. 7). From these reVol. 78, No. 2, 1975

Complete —Ammonium sulfate — Diketopimelate —Ammonium sulfate & diketopimelate —Ammonium sulfate & diketopimelate, + DPA (1.0 //moles)

Acid ninhydrin reaction

o-Aminobenzaldehyde reaction

(A420nm)

(A46Onm)

0.235 0.002 0.010

0.160 0.000 0.000

0.000

0.000

0.005

0.010

suits the product was tentatively concluded to be d' - piperidine - 2,6 - dicarboxylic acid. The color reaction with o-aminobenzaldehyde is specific for J'-piperidine, and J'-piperidine-2,6dicarboxylic acid is in equilibrium with a-ketoa'-aminopimelic acid, so this conclusion seems reasonable. The amount of 4'-piperidine-2,6dicarboxylic acid (tetra-hydrodipicolinic acid) calculated from o-aminobenzaldehyde reaction (Table I) was nearly equimolecular quantities with DPA. The configuration of the immediate precursor of DPA requires investigation. 1,4-Dihydrodipicolinic acid of Powell & Strange (2) seem to be the most likely isomeric forms in the system of diketopimelic acid and ammonia. Enzymatic Formation of Dipicolinic Acid from Diketopimelic Acid and Ammonia—The reaction of diketopimelic acid and ammonia is a very simple and convenient system for formation of DPA, in comparison with the system of pyruvate and aspartic /3 - semialdehyde. However, its physiological role is doubtful. The biological formation of this keto acid has not so far been observed in any microorganisms except for Penicillium citro-viride (#), and enzymatic formation of DPA has not been clearly demonstrated in this system. One reason for this is that the reaction can occur spontaneously. Cell free extract of Bacillus sub-

K. KIMURA and T. SASAKAWA

386

•5 3

l5

2 ..0

I

2

3

4

S

6

7

pH

8

FRACTION NUMBER

Fig. 7. Separation of the reaction products on a Sephadex G-10 column. The reaction mixture contained, in final volume of 1.0 ml, 50//moles of phosphate buffer, pH 6.4, 5 //moles of diketopimelate and 10 //moles of ammonium sulfate. Incubation was carried out at 37° for 60 min. Then 0.5 ml of the incubation mixture was subjected to gel-filtration on a Sephadex G-10 column (V0 = 15ml). 3 ml of each fraction were used for the following measurements: a, O, absorbance at 270nm; b, • , absorbance at 420nm (acid ninhydrin method); c, • , absorbance at 460 nm (o-aminobenzaldehyde method). The procedures are described in the " MATERIALS AND METHODS."

tills cause stimulation but their stimulatory effects could be explained by the presence of a suitable amount of ammonia and other possible factors, i.e. FMN and/or riboflavin in the extracts, and an adequate condition of pH in the resulting reaction mixture, as shown above. Another reason is that intermolecular oxidation and reduction of the intermediates may occur. DPA formation without oxygen uptake, described in the previous paper (7), might indicate the occurrence of a dismutation reaction. Accordingly, in this work, a purified preparation of dihydrodipicolinate reductase from Bacillus subtilis (5) was used under aerobic and anaerobic conditions to examine the enzymatic formation of DPA. Enzymatic Formation of DPA—Dihydrodipicolinate reductase catalyzes the reduction of dihydrodipicolinate, prepared from pyruvate

Fig. 8. Optimum pH for the catalytic effect of dihydrodipicolinate reductase. The reaction conditions were as for Fig. 3, except for the addition of 3.0 //g of the dihydrodipicolinate reductase (specific activity=30 //moles/min/mg protein). The amount of DPA was calculated from A277.5 —^274 • The enzymatic formation was calculated by subtraction of spontaneous formation ( • ) .

30

60

90

120

150

TIME (minutes)

Fig. 9. Catalytic effect of dihydrodipicolinate reductase. 3.0 ml of reaction mixture, in a quartz cell of 1.0 cm light path, contained 50 //moles of phosphate buffer, pH 6.4, 5 //moles of diketopimelate, 10 //moles of ammonium sulfate and 3.0 //g of the enzyme (reductase activity=30 //moles/min/mg protein) (curve 1), or the same amount of heated enzyme (5 min at 77-79°) (curve 2), or no enzyme (curve 3). Incubation was carried out at room temperature (21°) and absorption at 270 nm was measured.

and aspartic ^-semialdehyde, with NADPH as reductant. The reductase is a FMN-enzyme and free of non-heme iron, and it possesses diaphorase activity but not oxidase activity. / . Biochem.

DIPICOLINIC ACID FORMATION FROM DKP AND AMMONIA

However, it was found that this enzyme could oxidize dihydrodipicolinate formed from diketopimelic acid and ammonia, as shown in Fig. 8. The optimum pH of the enzymatic reaction was 6.4. The products separated from the reaction mixture by Sephadex G-10 gel filtration had the same UV-absorption spectrum of the Ca-dipicolinate complex, like the product of the nonenzymatic reaction. Figure 9 shows the sensitivity of the enzyme activity to heat-treatment for 5 min at 77-79°. Table II shows results on the coenzyme requirements of enzymatic reaction (a), and non-enzymatic reaction (b). On the enzymatic reaction, NAD+, NADP+, NADH, NADPH, and FMN were practically ineffective; while, on the nonenzymatic reaction, 10—1 urn FMN could reTABLE II. Effect of coenzymes on enzymatic and non-enzymatic formation of DPA. For the enzymatic reaction (a) the complete reaction system (1.0 ml) contained 50 /imoles of phosphate buffer, pH 6.4, 1.0 jumole of diketopimelic acid, 2.0 ^moles of ammonium sulfate, and 4 pig of enzyme. For the nonenzymatic reaction the reaction mixture was as for (a), except that enzyme was omitted. In both experiments, the effect of coenzymes were tested with indicated amounts. After incubation for 60 min at 37°, 0.5 ml of the reaction mixture was applied to a Sephadex G-10 column (K0 = 14ml). The amount of DPA was calculated from the absorbancy M277.5-A274) ( / ) . Reaction (a)

Enzymatic

Components

Complete + NAD+ (0.1 mM) + NADP+ (0.1 mM) + NADH (0.1 mM) - - - - + NADPH (0.1 MM) + FMN

(b)

Non-enzymatic

(10/IM)

Complete + FMN (10 HM) + FMN (1.0 ^M) + FeSO4 (1.0 mM) + FMN (1.0 fiM), FeSO4 (1.0 mM) + Boiled enzyme (4 fig)

Vol. 78, No. 2, 1975

387

place the enzyme. Effect of Oxygen on the DPA Formation in Spontaneous Reaction and Catalytic Reactions—Diketopimelic acid was incubated anaerobically with ammonia as described under "MATERIALS AND METHODS." The increase in UV-absorption at 270 nm was observed, indicating DPA formation, and the UV-absorption obtained under usual incubation conditions (aerobic, without flushing with air or oxygen) was essentially the same as that obtained under strictly anaerobic conditions. Similarly, the reaction rates were the same on anaerobic incubation in the presence of the enzyme (Fig. 10, A) or FMN (Fig. 11, A) as on aerobic incubation in the absence of the enzyme (Fig. 10, A) or FMN (Fig. 11, A ) . These results suggest that even under usual aerobic conditions no oxidation with oxygen leading to DPA formation occurs in the spontaneous reaction and DPA formation is due to dismutation of dihydrodipicolinic acid, perhaps resulting in tetrahydrodipicolinic acid. If this is the case, one would except that dihydrodipicolinic acid will be most difficult to isolate. Consequently oxygen is probably required for the catalytic action of the enzyme or FMN. These possibilities are verified by the results

DPA (^mole/ml) 0.28 0.22 0.23 0.24 0.21 0.24 0.081 0.22 0.13 0.12 0.16 0.10

120

180

240

TIME (minutes)

Fig. 10. Effect of dihydrodipicolinate reductase under usual (aerobic) or strictly anaerobic conditions. The reaction mixture was as for Fig. 9, except that 2.0 ^g of enzyme were used. Curves 1 (O) and 3 (A) correspond to curves 1 and 3, respectively, in Fig. 9. Curve 2 ( A ) : anaerobic incubation with enzyme. At the time indicated in the figure, the reaction mixture was exposed to air.

K. KIMURA and T. SASAKAWA

388

in Figs. 10 (curves 1 and 2) and 11 (curves 1 and 2). The enzyme (Fig. 10) and FMN (Fig. 11) clearly caused stimulation only under aerobic conditions. E

0.6

0.2

0

1 2

3

4

5

TIME (minutes)

TIME (minutes)

Fig. 11. Effect of FMN under aerobic or anaerobic conditions. The reaction mixture was as for Fig. 9, except that 1 nmole of FMN was used instead of the enzyme (Curve 1). Curve 2 : reaction mixture was incubated anaerobically with FMN and at the indicated time, was exposed to air. Curve 3 : reaction mixture was incubated aerobically without FMN.

Fig. 12. Reduction of FMN with diketopimelic acid and ammonia under anaerobic condition. 0.2 //mole of FMN, 5.0 ^moles of diketopimelic acid and 200 /imoles of phosphate buffer, pH 7.0 were placed into Thunberg-type quartz cuvettes of 1 cm path length and 10 ^moles of ammonium sulfate was placed into a side chamber, in final volume of 3.0 ml, and made anaerobic by successive evacuation and flushing with N 2 . Then ammonium sulfate in side chamber was added to the main reaction mixture and FMN reduction was followed by absorption measurement at 455 nm. After 4 min, the reaction mixture was flushed with air.

(A)

—llOminh— —02=0^-—

02=0

Fig. 13. Effect of the reductase and FMN on the oxidation of the intermediate formed from diketopimelic acid and ammonia. Oxygen uptake was measured by a Clark-type oxygen electrode at 37°. (A) The reaction mixture contained, in final volume of 5.0 ml, 250 jumoles of phosphate buffer, pH 6.4, 5 /imoles of diketopimelic acid, 10 /imoles of ammonium sulfate. At the time indicated in the figure (A), 0.5 mg of the reductase (specific activity = 1.0) was added. (B) The reaction mixture was as for (A) except that 0.1 //mole of FMN was added instead of the enzyme, and phosphate buffer of pH 7.0 was used. Effect of catalase on O2-uptake in each reaction was tested, adding 0.1 mg of crystalline catalase in each reaction mixture before addition of the enzyme or FMN.

/ . Biochem.

DIPICOLINIC ACID FORMATION FROM DKP AND AMMONIA

To confirm that the FMN was involved in the oxidation of dihydrodipicolinic acid, the changes in the absorption at 445 nm were determined under anaerobic conditions. As shown in Fig. 12, the absorption at 445 nm rapidly disappeared only when both diketopimelic acid and ammonium sulfate were added under anaerobic conditions, and reappeared on flushing with air. These results indicate that FMN acts as a hydrogen carrier from dihydrodipicolinic acid to oxygen. Decrease of absorption at 445 nm obeyed good first-order kinetics and the rate constant was calculated as about 0.56 min~' from the data in Fig. 12. To make sure that the oxidation mechanism of the intermediate (1,4-dihydrodipicolinate) formed from diketopimelic acid and ammonia, oxygen uptake was determined in the presence of the reductase or FMN. When the reductase or FMN were added to reaction mixture containing diketopimelic acid and ammonia, a dramatic increase in O2-uptake appeared without an lag time (Fig. 13). In both cases, the rate of (Vuptake were decreased to one-half in the presence of catalase. DISCUSSION In this work the net synthesis of DPA from diketopimelic acid and ammonia was clearly shown to proceed both non-enzymatically and enzymatically by the action of dihydrodipicolinate reductase of Bacillus subtilis. The apparent optimum pH of the spontaneous reaction was about 8.2 and ferrous sulfate, FMN, and riboflavin increased the reaction rate. On the other hand, an acidic pH favored the spontaneous formation of DPA from the condensation product of pyruvatewjthasparticsemialdehyde, and this reaction was stimulated by mercuric ion ( / ) . However, ferrous sulfate and FMN had no effect in this system. These differences suggest that the precursors of DPA in the two reactions are different. The precursors in the pyruvate-aspartic semialdehyde system were tentatively concluded to be 2,3- and 2,'5-dihydrodipolinic acids (1), while in the diketopimelic acid-ammonia system the most likely precursor was 1,4-dihydrodipicolinic acid (2). It was found that oxygen had no effect on the Vol. 78, No. 2, 1975

389

rate of spontaneous formation of DPA from diketopimelic acid and ammonia, whereas oxygen was essential for the catalytic actions of FMN and dihydrodipicolinate reductase. On the basis of these results, the reaction mechanisms of DPA formation are proposed to be as follows:

a)

Diketopimelic acid+ammonia > dihydrodipicolinic acid Spontaneous reaction (aerobic or anaerobic) 2 dihydrodipicolinic acid > DPA+tetrahydrodipicolinic acid

b) Catalytic reaction (aerobic, X=reductase or FMN) dihydrodipicolinic acid+X DPA+XH 2 XH 2 +O 2 >X+H 2 O 2 The enzymatic oxidation of dihydrodipicolinic acid in this system may be ascribed to a sort of non-specific catalysis by several flavoproteins. It seems possible that the oxidation reaction of dihydrodipicolinic acid catalyzed by dihydrodipicolinate reductase may have some physiological role in DPA formation in the pyruvate-aspartic semialdehyde system. The reductase requires two substrates, NADPH and dihydrodipicolinic acid, for activity. As shown in a previous paper (5), the reductase has FMN as a prosthetic group, and the hydrogen transfer pathway is considered to be NADPH—> FMN —»dihydrodipicolinate. Kinetic experiments on the binding of NADPH and dihydrodipicolinate (2,3- or 2, 5-) to the enzyme suggest that the reductase catalyzes the reduction of the dihydrodipicolinate by a ping pong mechanism (10). The reductase posseses diaphorase activity but not oxidase activity. However, in the presence of 0.5 raM of DPA, the reductase showed NADPH oxidase activity (unpublished data), and the dihydrodipicolinate (2,3- or 2,5) reducing activity of the reductase was inhibibited remarkably. Inhibition by DPA was competitive with NADPH and showed positive cooperativity (10). Thus, in the present work, the oxidation of 1,4-dihydrodipicolinate catalyzed by the reductase can be assumed to have occurred in the following way: the 1,4-dihydrcdipicolinate from a, e-diketo-

390

K. KIMURA and T. SASAKAWA

pimelate and ammonia acted as a hydrogen donor, instead of NADPH, and then the reduced form of FMN bound to the reductase was oxidized by oxygen. The conformational alterations of the reductase by DPA were of great interest in connection with sporulation, as a control mechanism of the lysine biosynthesis pathway and DPA formation in Bacillus subtilis. The authors thank Mr. Isao Mitome and Mr. Keiichiro Ishida for technical assistance. REFERENCES 1. Kimura, K. (1974) / . Biochem. 75, 961-967

2. Powell, J.F. & Strange, R.E. (1959) Nature 184, 878-880 3. Doi, R.H. & Halvorson, H.O. (1961) / . Bacteriol. 81, 642-648 4. Cope, A.C. & Fournier, A. (1957) / . Am. Chem. Soc. 79, 3896-3899 5. Kimura, K. (1975) / . Biochem. 77, 405-413 6. Basso, L.V., Rao, D.R., & Ridwell, V.W. (1962) / . Bio/. Chem. 237, 2239-2245 7. Farkas, W. & Gilvarg, C. (1965) / . Biol. Chem. 240, 4717-4722 8. Tanenbaum, S.W. & Kaneo, K. (1964) Biochemistry 3, 1314-1322 9. Koepsell, H.J. & Sharpe, E.S. (1952) Arch. Biochem. Biophys. 38, 443-449 10. Kimura, K. & Goto, T. (1975) / . Biochem. 77, 415-420

/ . Biochem.

Pyridine-2, 6-dicarboxylic acid (dipicolinic acid) formation in Bacillus subtilis. II Non-enzymatic and enzymatic formations of dipicolinic acid from alpha, epsilon-diketopimelic acid and ammonia.

/ . Biochem., 78, 381-390 (1975) Pyridine-2, 6-dicarboxylic Acid (Dipicolinic Acid) Formation in Bacillus subtilis II. Non-enzymatic and Enzymatic Fo...
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