O Succinylbenzoic acid

Menaquinone. The incorporation of [2- C]mevalonate and [2- C]-2-methyl-l,4-naphthoquinone into MK-4, normally considered a bacterial quinone, has been demonstrated in marine invertebrates such as crabs and starfish." Incorporation into 2,3-epoxy-MK-4 (163) was also observed. Cell-free extracts have been prepared from Escherichia coli which catalyse the conversion of o-succinylbenzoic acid (164) into l,4-dihydroxy-2-naphthoic acid (165) and menaquinones. In the presence of farnesyl pyrophosphate the major menaquinone produced was MK-3. Genetic studies with mutants of E. coli K12 that require (164) offer support for the generally accepted pathway for MK biosynthesis via (164) and (165)." The enzyme system that catalyses the attachment of the polyprenyl side-chain to 1,4-dihydroxy-2-naphthoic acid to form demethylmenaquinone-9 (166) has been isolated from E. colU ... 

The biosynthesis of menaquinone is outlined in Fig. 44. Isomerization of chorismate to isochorismate followed by condensation with a-ketogluta-rate and aromatization gives o-succinylbenzoic acid. Conversion of 238 to the CoA thio ester, followed by cyclization, prenylation and methylation completes the biosynthesis. The biosynthesis of the prenyl side chain follows the alternative terpene biosynthetic pathway described for ubiquinone. 

The biosynthesis of these anthraquinones parallels those of the menoquinones in bacteria and naphtoquinones of plants for example juglone, vitamin K and lawsone. These compounds are also derived from shikimic (or chorismic) and a-ketoglutaric acids via o-succinylbenzoic acid [5,24]. 1,4-Dihydroxy-2-naphtoic acid is the branching point in the biosynthesis of menoquinones, naphtoquinones and anthraquinones [4],... 

The biosynthesis of menaquinones in E. coli (Fig. 5) starts with the conversion of isochorismic acid and a-ketoglutaric acid in the presence of thiamine pyrophosphate [105-107] to 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) catalyzed by SHCHC synthase, and a-ketoglutarate decarboxylase, both encoded by the menD gene [104,108,109]. SHCHC is converted to o-succinylbenzoic acid by dehydration, catalyzed by a protein encoded by menC (Table 1) [110]. Palaniappan et al., [111] showed for the first time the biosynthesis of menaquinones via o-succinyl benzoic acid in B. subtilis including the activity of the enzymes. 

The key intermediate in the formation of these compounds is o-succinylbenzoic acid (OSB) (38) which is formed from wo-chorismic acid and a-ketoglutararic acid (39), a tticarboxylic acid cycle intermediate (Fig. 6.7) (Si-mantiras and Leistner, 1989). a-Ketoglutaric acid can be converted to glutamic acid, which previously was proposed... 

Formation of the coenzyme A ester of the aliphatic carboxyl group of o-succinylbenzoic acid (38) appears to activate the methylene proton sufficiently to permit attack at the aromatic carboxyl group (Fig. 6.7). 2-Carboxy-4-oxotetra-lone (COT) (43) and/or l,4-dihydroxy-2-naphthoic acid (DHNA) (40) are likely intermediates for later stages of synthesis. The enzymatic conversion to DHNA with naphthoate synthase (which consists of OSB CoA synthetase and DHNA synthetase) requires ATP, Mg ", and coenzyme A. 1,4-Dihy-droxy-2-naphthoic acid (40) is a precursor of vitamin K2 (35) and menaquinones (such as 44) (Inouye and Leistner,... 

Heide, L., Arendt, S., Leistner, E. Enzymatic synthesis, characterization, and metabolism of the coenzyme A ester of o-succinylbenzoic acid, an intermediate in menaquinone (vitamin K2) biosynthesis. J. Biol. Chem. 257, 7396-7400 (1982)... 

A rather unique branch of the shikimate pathway operates in the biosynthesis of naphthoquinones related to vitamin K (menaquinone) (Fig. 30). Seven of the ten carbon atoms of the naphthoquinone ring system are derived from the seven carbon atoms of shikimic acid. The remaining three carbons are provided by the three center carbon atoms of a-ketoglutaric acid in a reaction leading to the unique intermediate o-succinylbenzoic acid. Cyclization of the latter produces the intermediate 1,4-dihydroxynaphthoic acid, the substrate for an isoprenylation reaction which occurs with simultaneous loss of COa Methylation of the 3 position then completes the reaction sequence. Studies by Meganathan and Bentley had indicated that chorismic acid is the substrate for the thiamine pyrophosphate-... 

Fig. 31. Formation of o-succinylbenzoic acid from iso-chorismic acid. Reprinted from Reference 53 with permission of Pergamon Press Ltd.

Site of activation of o-succinylbenzoic acid during its conversion to menaquinones (vitamin Ka). FEES Lett. 137 53-56. 

In agreement with the assumption > that chorismic acid is the branch point, it was postulated that a cell free system catalyzed the conversion of chorismic acid and a-ketoglutaric acid (III) to o-succinylbenzoic acid (II) Later it became evident, however, that the chorismic acid sample employed in these experiments contained trace amounts of iso-chorismic acid (I). Indeed, iso-chorismic acid (I) is converted to -succinylbenzoic acid (II) in the presence of a-ketoglutaric acid (III) in almost 90% yield. ... 

The enzymic conversion of o-succinylbenzoic acid (II) to 1,4-dihydroxy-2-naphthoic acid (DHNA) (V) is a reaction dependent on ATP, Mg + and coenz3nne A. The product DHNA is a precursor of vitamin K2. One may assume that both carboxyl groups in OSB (II) are activated during the conversion to DHNA (V). Activation of the "aliphatic" group would provide for the acidity of a proton on the methylene group... 

When this mixture is treated with alkali for short time the thioester bonds hydrolyze first. Thus methylesters (X, XI) of -succinylbenzoic acid are formed. Moreover, we found that the aromatic methylester (XI) is more labile in alkali than the aliphatic methylester (X). Hence in the experimental sequence depicted in Figure 3, one ends up with the aliphatic methylester of o-succinylbenzoic acid (X) and concludes that it is the aromatic carboxyl group which is activated. As we now know, only part of this conclusion is correct because both the aliphatic (VI) and the aromatic (VII) ester are formed enzymically at pH 7.9. But only the aliphatic ester (VI) is converted to DHNA (V) by naphthoatsynthase. It should be noted that biosynthetic schemes shown in previous publications are in error a more recent one is correct. 

Vitamin K with only one prenyl unit (XVI) has been isolated from callus tissue of Catalpa ovata. This naphthoquinone is associated with l-hydroxy-2-methylanthraqui-none (XVII) and o succinylbenzoic acid (II) is a precursor of these compounds. During their biosynthesis 2-prenyl-carboxyoxotetralone (XVIII) and catalponone (XIX) are formed. This was established by trapping labelled XVIII and XIX after administration of radioactive o-succinyl-benzoic acid (II) to ovata tissue cultures. The chirality of XVIII and XIX was shown to be 2S (XVIII) and 2R (XIX) respectively. Formation of catalponone (XIX) does not proceed through DHNA (V). The biosynthesis of anthra-quinone in the tissue culture of iC. ovata may therefore be different from the cultures of Rubia, Morinda and Galium in that carboxyoxotetralone (XX) is prenylated instead of... 

Biochemical and molecular information on the juglone biosynthetic pathway is lacking. While many labeled precursors, such as glucose, acetate and shikimic acid, were successfully incorporated in juglone, o-succinylbenzoic acid appears to be the immediate precursor of the naphthoquinone [100], which is a key step in the formation of more oxidized naphthoquinones [101]. 

Leduc, Dansette and Azerad ° and Bentley and his collaborators have found o-succinylbenzoic acid (39) specifically labelled with to be efficiently incorporated into bacterial menaquinones. However, the subsequent stages in the biosynthetic pathway are not clear and they have been the subject of confficting views. Thus Leistner,... 

However, the more recent work of Leistner on the biosynthesis of alizarin (101) in Rubia tinctora, although it has confirmed the position of o-succinylbenzoic acid (108) and L-glutamic acid (110) as intermediates, has cast doubts on the validity of some aspects of the proposed pathway [Figure 6.14) and in particular the role of 1,4-naphthoquinone (109). It was thus noted by Leistner that using 7-[ C]-(—)-shikimic acid (107) as substrate the radioactive label was incorporated directly into C-9 of the alizarin molecule whilst 2-[ C]-L-glutamic acid (110) labelled C-10 specifically. These... 

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