glucose and 18 ,uM menadione and grown overnight. For large-scale growth, a loopful of inoculum from plate cultures was inoculated into 5 ml of liquid medium ...
JOURNAL OF BACrERIOLOGY, May 1994, p. 2648-2653
Vol. 176, No. 9
0021-9193/94/$04.00+0 Copyright © 1994, American Society for Microbiology
Biosynthesis of o-Succinylbenzoic Acid in Bacillus subtilis: Identification of menD Mutants and Evidence against the Involvement of the a-Ketoglutarate Dehydrogenase Complex C. PALANIAPPAN,1 H. TABER,2 AND R. MEGANATHANl* Department of Biological Sciences, Northem Illinois University, DeKalb, Illinois 60115,1 and Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 122012 Received 30 September 1993/Accepted 28 February 1994
The biosynthesis of o-succinylbenzoic acid (OSB), the first aromatic intermediate involved in the biosynthesis of menaquinone (vitamin K2) is demonstrated for the first time in the gram-positive bacterium Bacilus subtilis. Cell extracts were found to contain isochorismate synthase, 2-succinyl-6-hydroxy-2,4-cyclohexadiene1-carboxylic acid (SHCHC) synthase-a-ketoglutarate decarboxylase and o-succinylbenzoic acid synthase activities. An odhA mutant which lacks the decarboxylase component (usually termed El, EC 1.2.4.2, oxoglutarate dehydrogenase [lipoamide]) of the a-ketoglutarate dehydrogenase complex was found to synthesize SHCHC and form succinic semialdehyde-thiamine pyrophosphate. Thus, the presence of an alternate G-ketoglutarate decarboxylase activity specifically involved in menaquinone biosynthesis is established for B. subtilis. A number of OSB-requiring mutants were also assayed for the presence of the various enzymes involved in the biosynthesis of OSB. All mutants were found to lack only the SHCHC synthase activity.
adione (Sigma Chemical Co.) at - 80°C. Cells from frozen cultures were streaked on L agar plates containing 0.5% glucose and 18 ,uM menadione and grown overnight. For large-scale growth, a loopful of inoculum from plate cultures was inoculated into 5 ml of liquid medium of the same composition contained in test tubes and grown for 3 h with shaking at 37°C. The tube cultures were inoculated into 50 ml of medium in 250-ml Erlenmeyer flasks and grown for an additional 3 h. These cultures were then inoculated into 500 ml of medium contained in 1-liter flasks, and shaking continued for an additional 6 to 7 h until the culture reached a Klett reading of 150 at 600 nm. During the growth of men mutants, reversion was monitored by plating on L agar. Harvesting of cells and preparation of cell extracts. Cells were harvested by centrifugation at 4,000 x g for 10 min. Cell extracts were prepared as described by Meganathan et al. (12), except that 0.15 mM phenylmethanesulfonyl fluoride was included in the buffer. The supernatant obtained was used in enzyme assays. In some experiments, the extract was treated with a 2% solution of protamine sulfate until the volume of the extract increased by 30% as described elsewhere (17), and the precipitate formed was removed by centrifugation at 17,000 x g for 15 min. The cleared supernatant was used in enzyme assays. The protein content of cell extracts was determined by the procedure of Bradford (3). Enzyme assays. We performed assays for ot-ketoglutarate dehydrogenase (KGDH), isochorismate synthase, SHCHC synthase, KDC, and OSB as described below. KGDH. The El component of the KGDH complex was assayed according to the method of Reed and Willms (20, 21). Isochorismate synthase. The isochorismate synthase assay was performed exactly as described by Popp et al. (19), in a total volume of 1.0 ml. The quantity of enzymatically synthesized isochorismate was determined by thermal conversion to salicylate (27). The salicylate formed was assayed spectrophotofluorimetrically in a Perkin-Elmer LS3 fluorescence spectrometer using an excitation wavelength of 300 nm and an emission wavelength of 410 nm. The amount of salicylate was
The menaquinone (vitamin K2) biosynthetic pathway has been extensively studied and was recently reviewed (2, 25). The first committed step in menaquinone biosynthesis is the formation of the prearomatic compound 2-succinyl-6-hydroxy2,4-cyclohexadiene-1-carboxylic acid (SHCHC) (6). SHCHC is formed from isochorismate and ct-ketoglutarate by the action of SHCHC synthase and a-ketoglutarate decarboxylase (KDC) in a thiamine pyrophosphate (TPP)-dependent reaction (11, 17, 18). In Escherichia coli, it has been shown that both SHCHC synthase and KDC activities are encoded by the menD gene (17). The bifunctional nature of this enzyme has also been recently confirmed by purification of the enzyme to homogeneity (16). The SHCHC from the reaction is subsequently aromatized with the loss of a hydroxyl group to o-succinylbenzoic acid (OSB) by the enzyme OSB synthase
(Fig. 1) (17, 18).
The further conversion of the benzenoid OSB to the naphthalenoid 1,4-dihydroxy-2-naphthoic acid has been shown in E. coli (23) and a number of gram-positive organisms (10, 12, 13). In contrast, the formation of OSB from isochorismate and a-ketoglutarate has been shown only in E. coli. Earlier attempts to demonstrate the formation of OSB in the grampositive bacterium Bacillus subtilis met with uniformly negative results. However, studies with E. coli led to a better understanding of the reactions, refinement of assay procedures, and the availability of amplified men genes (17, 18, 20). With these resources in hand, we undertook an examination of OSB formation and demonstrate for the first time the biosynthesis of OSB in cell extracts of B. subtilis.
MATERUILS AND METHODS Bacterial strains and growth conditions. The parent strain (men') of B. subtilis, the mutant strains, and their genotypes are described in Table 1. Cultures were routinely stored in glycerated L broth containing 0.5% glucose and 18 ,uM men*
Corresponding author. Phone: (815) 753-7803. Fax: (815) 753-
0461. 2648
MENAQUINONE (VITAMIN K2) BIOSYNTHESIS
VOL. 176, 1994 OH HOOC -%,,0
Chorismate
COOH
1
1
2- Ketoglutarate 2
OH
1
L;u2
-
0 HOOC 0u,,,,
+ TPP -
COOH
I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ e9
CH2
-CH2CH COOH
OH
Isochorismate 3
OH
COCCOH O
C SHCHC
O
4
200 Rl contained in a stoppered reaction vessel (15). The reaction was carried out at 37°C for 30 min. The "4CO2 released was trapped, and the radioactivity was determined. The procedure for trapping and counting of "4Co2 is as described by Mitchell et al. (15), with the following modifications. In the plastic cups suspended from the stopper of the reaction flask, the methylbenzethonium hydroxide solution was replaced by a folded filter paper disk (2 by 1.5 cm) containing 50 RI of 30% KOH to capture the released CO2. After absorption of the 14C02, the filter paper disk was transferred to mini vials containing 5 ml of Econosafe Scintillation fluid (Research Products International) and the radioactivity was measured. OSB synthase assay. The OSB synthase assay was carried out as described by Popp et al. (19) for overall OSB synthesis, except that the isochorismate was replaced by 277 nmol of chorismate and carried out in a total volume of 300 ,l. The OSB formed was measured by HPLC (19). RESULTS
H~OO
0
~H20
COOH COOH
OSB 0
FIG. 1. Pathway for the biosynthesis of OSB. 1, isochorismate synthase; 2, KDC; 3, SHCHC synthase; 4, OSB synthase.
determined by reference to a standard curve, and the amount of isochorismate formed was calculated (19, 27). SHCHC synthase. The SHCHC synthase assay mixture was identical to that described by Popp et al. (19) except that the reaction was carried out in a total volume of 300 RI and the isochorismate was replaced by 277 nmol of chorismate. The product of the reaction, SHCHC, was assayed in two ways. (i) The SHCHC formed was converted to OSB by alkaline hydrolysis and assayed by high-performance liquid chromatography (HPLC) (19). Authentic OSB samples were run as standards. (ii) The SHCHC formed was converted to OSB enzymatically by using a cell extract from E. coli RM862 which produces amplified levels of OSB synthase. The OSB formed was assayed by HPLC as described above. KDC. The KDC reaction mixture contained 0.5 ,uCi of 2[U-14C]ketoglutarate and other components as described by Palaniappan et al. (17). The products of the reaction 2[U4C]succinic semialdehyde and "4CO2 were assayed as follows. (i) Assay of succinic semialdehyde. The 2[U-14C]succinic semialdehyde was converted to its dinitrophenylhydrazone derivative and separated from the ao-ketoglutarate dinitrophenylhydrazone by using thin-layer chromatography as described previously (17). The thin-layer chromatograms were scanned for radioactivity with a Packard model 7201 radiochromatogram scanner.
(ii) Assay of "'CO2 release. The incubation mixture consisted of 10 p.mol of Tris-HCl buffer (pH 8.0), 0.13 ,umol of TPP, and 0.5 ,uCi of 2[U-14C]ketoglutarate in a total volume of
2649
Effects of pH on the formation of isochorismate, SHCHC, and OSB. Since the formation of OSB from chorismate involves isochorismate and SHCHC as intermediates, it was important to study the formation of these compounds individually at various pH levels. Accordingly, cell extracts from the men+ strain RB1 were incubated with chorismate at pH 7, 8, and 9 and the formation of isochorismate was measured. As seen from Table 2, for the conditions tested, the largest amount of isochorismate formation occurred at a pH of 9.0. For measurement of SHCHC and OSB, cell extracts were incubated with chorismate, a-ketoglutarate, and TPP at the three pH levels and the products were measured from separate incubation mixtures. The formation of both SHCHC and OSB occurred optimally at pH 8.0 (Table 2). Comparison of enzyme activities in crude and protamine sulfate-treated extracts. During studies on E. coli SHCHC synthase-KDC, it was discovered that protamine sulfate treatment resulted in partial purification of the enzyme. The B. subtilis extract was subjected to protamine sulfate treatment TABLE 1. Bacterial strains used in this study Strain
B. subtilis RB1 RB949
RB321 RB386 RB392 RB395 RB403 RB438 E. coli JRG862
RM862 TK3D18 RM18 Plasmids pMS29
pJP1O1
Relevant genotype
men + trpC2
Reference or source
men+ metC3 glyB133
BGSCa A. L. Sonenshein
odhA109 men321 trpC2 men318 trpC2 men322 trpC2 men311 trpC2 men315 trpC2 men317 trpC2
26 26 26 26 26 26
gal trpA trpR iclR rpsL menCl JRG862/pMS29
7
TK3D18/pJP101
22 W. Epstein 16
menC+ menD+
22 18
A(kdp-suc)D18 A(gal-bio)
a Bacillus Genetic Stock Center, Ohio State University, Columbus, Ohio.
2650
PALANIAPPAN ET AL.
J. BAcrERIOL.
TABLE 2. Effects of pH on the accumulation of isochorismate, SHCHC, and OSB in extractse from the men' strain RB1
60
Accumulation (nmol)
50
SHCHC
pH
OSB
Isochorismate
7.0 8.0 9.0
22.4 30.1 31.5
-
55
Hydrolyticb
Enzymaticc
9.8 24.5 22.5
8.1 22.8 20.8
, 45
E0
40
4.2 10.1 6.2
a For each assay, 7.0 mg of crude extract was incubated for 1 h as described in Materials and Methods and the accumulated products were measured. b The SHCHC accumulated was converted to OSB by the alkaline hydrolysis method and assayed by HPLC. c The SHCHC accumulated was converted to OSB by using a cell extract (400 p,g of protein) of E. coli RM862, which produces amplified levels of OSB synthase, and assayed by HPLC.
-
0
35
/
30
/
01
25
-
20 o0 -
0
15 0
10 5
and assayed for the activity of isochorismate synthase, SHCHC synthase-KDC, and OSB synthase at a pH of 8.0. The results are presented in Table 3. The isochorismate synthase specific activity increased by 3.5-fold, and the SHCHC synthase-KDC and OSB synthase specific activities increased approximately 2-fold in the protamine sulfate-treated supernatant fraction. Time course of formation of isochorismate, SHCHC, and OSB. Isochorismate and SHCHC are highly unstable intermediates which must be generated in vitro. Thus, for the assay of each enzyme involved in the sequence of reactions leading to the formation of OSB (Fig. 1), it was important to generate a sufficient accumulation of substrate(s) in the reaction mixture. In other words, the enzyme of interest should be the only rate-limiting reaction in the sequence of reactions. Hence, it was important to determine the quantity of product(s) accumulated over timed intervals. With this objective in mind, protamine sulfate supernatant was incubated with chorismate and the amount of isochorismate accumulated was determined at 30-min intervals for up to 150 min. For the determination of SHCHC accumulation, an equal amount of protamine sulfate supernatant was incubated with chorismate, oL-ketoglutarate, and TPP and the amount of SHCHC formed was assayed after alkaline hydrolysis over the same timed intervals. Similarly, the protamine sulfate supernatant was incubated with chorismate, ot-ketoglutarate, and TPP and the OSB formed was determined. As shown in Fig. 2, isochorismate showed the largest accumulation, followed by SHCHC, and OSB showed the least accumulation. Conversely, in the sequence of reactions leading to the formation of OSB (Fig. 1), isochorismate synthase is the most active enzyme, followed by SHCHC synthase-KDC, with OSB synthase being the least active. Thus, under the conditions used for the assays, there is sufficient (nonlimiting) accumulation of isochorismate in the assay mixture to facilitate TABLE 3. Comparison of enzymatic activities in crude and protamine sulfate-treated cell extracts from the men' strain RB1 Isochorismate SHCHC synthaseOSB synthase KDC synthase
Extract'
Crude Prot. S04
nmol/h
Sp actb
nmol/hc
Sp act
nmol/h
Sp act
12.1 20.0
3.7 14.7
11.2 9.4
3.5 6.3
2.1 1.7
0.7 1.1
a For the assay, 3.25 mg of protein was used for the crude extract and 1.5 mg of protein for the protamine sulfate-treated extract. b Specific activity is expressed as nanomoles per hour per milligram of protein. SHCHC was assayed after alkaline hydrolysis.
0 0
30
60
90
120
150
180
Time (mln)
FIG. 2. Time course of formation of isochorismate, SHCHC, and OSB. Each incubation mixture contained 3.0 mg of protein from a protamine sulfate-treated supernatant. *, isochorismate; 0, SHCHC; 0, OSB. Details of the assays are described in Materials and Methods.
the assay of SHCHC synthase and there is sufficient accumulation of SHCHC for OSB synthase assay. Evidence against the involvement of KGDH complex. The first committed step in the menaquinone biosynthetic pathway involves the addition to isochorismate of the succinic semialdehyde-TPP anion formed via a reaction similar to that catalyzed by the El component of the KGDH complex (2). In E. coli, it has been demonstrated that an enzymatic activity distinct from the KGDH complex is required for the KDC reaction (9, 17). It was important to establish whether a KDC activity distinct from the El activity of KGDH complex was also involved in B. subtilis. In previous studies with E. coli, it was demonstrated that treatment with protamine sulfate results in the precipitation of KGDH, while the KDC activity remains in the supernatant (17). Hence, a cell extract from B. subtilis and the protamine sulfate supernatant were assayed for the El activity of the KGDH complex. The cell extract from the wild-type strain showed an El specific activity of 612 nmol/h/mg of protein. In contrast, the protamine sulfate supernatant failed to show any El activity. However, the protamine sulfate supernatant showed KDC activity, as determined by the formation of [14C]succinic semialdehyde (Fig. 3A) and by the release of 14C02 (Table 4) from [14C]ot-ketoglutarate. To further confirm the absence of a role of the KGDH complex in menaquinone biosynthesis, an odhA mutant was assayed for El and as expected was found to lack the enzyme activity. However, when the odh/A mutant was assayed for KDC activity, it formed approximately the same amount of ["4C]succinic semialdehyde (Fig. 3B) and 14CO2 (Table 4) from [14C]ox-ketoglutarate as the protamine sulfate supernatant from the wild-type strain. These results clearly establish that the KDC activity involved in menaquinone biosynthesis is distinct from that of the El of the KGDH complex. Enzymatic analysis of OSB-requiring mutants. Mutants of B. subtilis blocked in the menaquinone biosynthesis pathway are unable to grow on either synthetic or complex media unless supplied with the menaquinone analog menadione. Hence, it was important to establish that the enzymes are not repressed
MENAQUINONE (VITAMIN K2) BIOSYNTHESIS
VOL. 176, 1994 TABLE 4. Activities of various enzymes in protamine sulfatetreated extracts of men', odhA, and OSB-requiring men mutants
2651
100
Formation in the
StraiIsochorismate synthasea
SHCHC
synthasea synthasea SHCHC synthase-KDC SHCHCC OSB syntased
11,624 11,000 44,406 32,512 26,560 21,548 24,356 52,600
14.7 9.0 11.6 16.2 14.7 8.0 7.3 12.0
RB1 RB949 RB321 RB386 RB392 RB395 RB403 RB438
presence of E. coli
OSB
7.0 6.5
