The Corynebacterianeae such as Corynebacterium glutamicum and Mycobacterium tuberculosis possess several unique and structurally diverse lipids, including the genus-specific mycolic acids. Although the function of a number of genes involved in fatty acid and mycolic acid biosynthesis is known, information relevant to the initial steps within these biosynthetic pathways is relatively sparse. Interestingly, the genomes of Corynebacterianeae possess a high number of accD genes, whose gene products resemble the -subunit of the acetyl-CoA carboxylase of Escherichia coli, providing the activated intermediate for fatty acid synthesis. We present here our studies on four putative accD genes found in C. glutamicum. Although growth of the accD4 mutant remained unchanged, growth of the accD1 mutant was strongly impaired and partially recovered by the addition of exogenous oleic acid. Overexpression of accD1 and accBC, encoding the carboxylase ␣-subunit, resulted in an 8-fold increase in malonyl-CoA formation from acetyl-CoA in cell lysates, providing evidence that accD1 encodes a carboxyltransferase involved in the biosynthesis of malonyl-CoA. Interestingly, fatty acid profiles remained unchanged in both our accD2 and accD3 mutants, but a complete loss of mycolic acids, either as organic extractable trehalose and glucose mycolates or as cell wall-bound mycolates, was observed. These two carboxyltransferases are also retained in all Corynebacterianeae, including Mycobacterium leprae, constituting two distinct groups of orthologs. Furthermore, carboxyl fixation assays, as well as a study of a Cg-pks deletion mutant, led us to conclude that accD2 and accD3 are key to mycolic acid biosynthesis, thus providing a carboxylated intermediate during condensation of the mero-chain and ␣-branch directed by the pks-encoded polyketide synthase. This study illustrates that the high number of accD paralogs have evolved to represent specific variations on the well known basic theme of providing carboxylated intermediates in lipid biosynthesis.
The suborder Corynebacterianeae comprises bacteria like Mycobacterium tuberculosis and Corynebacterium glutamicum, and these bacteria contain in addition to the linear fatty acids, unique ␣-branched -hydroxy fatty acids, called mycolic acids. Whereas acetyl-coenzyme A (CoA) carboxylase activity is required to provide malonyl-CoA for fatty acid synthesis, a new type of carboxylase is apparently additionally present in these bacteria. It activates the ␣-carbon of a linear fatty acid by carboxylation, thus enabling its decarboxylative condensation with a second fatty acid to afford mycolic acid synthesis. We now show that the acetyl-CoA carboxylase of C. glutamicum consists of the biotinylated ␣-subunit AccBC, the -subunit AccD1, and the small peptide AccE of 8.9 kDa, forming an active complex of approximately 812,000 Da. The carboxylase involved in mycolic acid synthesis is made up of the two highly similar -subunits AccD2 and AccD3 and of AccBC and AccE, the latter two identical to the subunits of the acetyl-CoA carboxylase complex. Since AccD2 and AccD3 orthologues are present in all Corynebacterianeae, these polypeptides are vital for mycolic acid synthesis forming the unique hydrophobic outer layer of these bacteria, and we speculate that the two -subunits present serve to lend specificity to this unique large multienzyme complex.
Reversible 2,6-dihydroxybenzoate decarboxylase from Rhizobium sp. strain MTP-10005 belongs to a nonoxidative decarboxylase family. We have determined the structures of the following three forms of the enzyme: the native form, the complex with the true substrate (2,6-dihydroxybenzoate), and the complex with 2,3-dihydroxybenzaldehyde at 1.7-, 1.9-, and 1.7-Å resolution, respectively. The enzyme exists as a tetramer, and the subunit consists of one (␣) 8 and is assumed to be the catalytic base. On the basis of the geometrical consideration, substrate specificity is uncovered, and the catalytic mechanism is proposed for the novel Zn 2؉ -dependent decarboxylation.The nonoxidative decarboxylation catalyzed by decarboxylases such as 2,3-dihydroxybenzoate (1-5), 2,5-dihydroxybenzoate (6), 3,4-dihydroxybenzoate (7), 4,5-dihydroxyphthalate (8 -10), and 4-hydroxybenzoate decarboxylase (11, 12) is a poorly understood reaction. These enzymes have been reported to require neither a cofactor such as NAD ϩ , pyridoxal 5Ј-phosphate, or thiamine monophosphate nor a pyruvoyl group for catalytic activity. In studies on these enzymes, the interest is focused on their substrate specificities and catalytic mechanisms.We isolated a thermophilic reversible 2,6-dihydroxybenzoate (␥-resorcylate) decarboxylase (GRDC) 2 from Rhizobium sp. strain MTP-10005 and characterized it (13). The GRDC catalyzes the decarboxylation of 2,6-and 2,3-dihydroxybenzoate to 1,3-dihydroxybenzene (resorcinol) and 1,2-dihydroxybenzene, respectively but does not act on 2,4-, 2,5-, 3,4-, 3,5-dihydroxybenzoate, 2-hydroxybenzoate, or 3-hydroxybenzoate (Scheme 1) . 2,6-Dihydroxybenzoate is an important intermediate of medicine and agricultural or industrial chemicals (14 -16). However, it is generated together with 2,4-dihydroxybenzoate as a by-product at a rate of about half and half by traditional chemical methods (17). 2,6-Dihydroxybenzoate is expected to be produced specifically from 2,6-dihydroxybenzene by the reverse carboxyl reaction of GRDC.Recently, Ishii et al. (18) reported the purification and characterization of GRDC from Rhizobium radiobacter WU-0108, Agrobacterium tumefaciens IAM12048 (19), and Pandoraea sp. 12B-2 (20). They reported that these enzymes also catalyze the reversible decarboxylation of 2,6-dihydroxybenzoate without cofactors and have a similar substrate specificity. Orotidine 5Ј-monophosphate decarboxylase catalyzes the cofactor-independent decarboxylation. On the basis of the x-ray structure of the enzyme, it is proposed that the decarboxylation of orotidine 5Ј-monophosphate proceeds by an electrophilic substitution mechanism in which decarboxylation and carbon-carbon bond protonation by Lys 62 occur in a concerted way (21,22). To elucidate the overall and active-site structure, the substrate recognition, and the reaction mechanism, we have determined the crystal structures of GRDC from the Rhizobium sp. strain MTP-10005 in the native form, GRDC complexed with the substrate 2,6-dihydroxybenzoate, and GRDC complexed with substrate anal...
In Escherichia coli, the enzyme called cysteine desulfhydrase (CD), which is responsible for L-cysteine degradation, was investigated by native-PAGE and CD activity staining of crude cell extracts. Analyses with gene-disrupted mutants showed that CD activity resulted from two enzymes: tryptophanase (TNase) encoded by tnaA and cystathionine beta-lyase (CBL) encoded by metC. It was also found that TNase synthesis was induced by the presence of L-cysteine. The tnaA and metC mutants transformed with the plasmid containing the gene for feedback-insensitive serine acetyltransferase exhibited higher L-cysteine productivity than the wild-type strain carrying the same plasmid. These results indicated that TNase and CBL did act on L-cysteine degradation in E. coli cells.
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