Cyclization mechanism catalyzed by an ATP-grasp enzyme essential for d-cycloserine biosynthesis.

In the biosynthetic pathway of an antitubercular antibiotic d-cycloserine (d-CS), O-ureido-d-serine (d-OUS) is converted to d-CS. We have previously demonstrated that DcsG, classified into the ATP-grasp superfamily enzyme, catalyzes the ring formation to generate d-CS, which is accompanied by the cleavage of a bond in the urea moiety of d-OUS to remove a carbamoyl group. Although the general ATP-grasp enzymes catalyze an ATP-dependent ligation reaction between two substrates, DcsG catalyzes specifically the generation of an intramolecular covalent bond. In the present study, cyanate was found in the reaction mixture, suggesting that carbamoyl group is eliminated as an isocyanic acid during the reaction. By the crystallographic and mutational investigations of DcsG, we anticipate the residues necessary for the binding of d-OUS. An acylphosphate intermediate must be bound at the narrow pocket of DcsG in a folded conformation, inducing the bond cleavage and the new bond formation to generate cyanate and d-CS, respectively. DATABASE: Structural data are available in Protein Data Bank database under the accession number 6JIL.

The structural and mutational analyses of O-ureido-L-serine synthase necessary for D-cycloserine biosynthesis.

UNLABELLED: We have recently been successful in cloning a gene cluster necessary for the biosynthesis of D-cycloserine (D-CS) from D-CS-producing Streptomyces lavendulae ATCC11924. Although dcsD, one of the ORFs located on the gene cluster, encodes a protein homologous to O-acetylserine sulfhydrylase that synthesizes L-cysteine using O-acetyl-L-serine together with sulfide, it functions to form O-ureido-L-serine as a D-CS biosynthetic intermediate, using O-acetyl-L-serine together with hydroxyurea (HU). In the present study, using crystallographic and mutational studies, three amino acid residues in DcsD that are important for the substrate preference toward HU were determined. We showed that two of the three residues are important for the binding of HU into the substrate-binding pocket. The other residue contributes to the formation of a loose hydrogen-bond network during the catalytic reaction. Information regarding the amino acid residues will be very useful in the design of a new catalyst for synthesizing the ß-substituted-L-alanine derivatives. DATABASE: The atomic coordinates and structure factors of wild-type DcsD and l-OUS-bound K43A mutant of DcsD have been deposited in the Protein Data Bank under accession codes 3X43 and 3X44, respectively.

Establishment of an in vitro D-cycloserine-synthesizing system by using O-ureido-L-serine synthase and D-cycloserine synthetase found in the biosynthetic pathway.

We have recently cloned a DNA fragment containing a gene cluster that is responsible for the biosynthesis of an antituberculosis antibiotic, D-cycloserine. The gene cluster is composed of 10 open reading frames, designated dcsA to dcsJ. Judging from the sequence similarity between each putative gene product and known proteins, DcsC, which displays high homology to diaminopimelate epimerase, may catalyze the racemization of O-ureidoserine. DcsD is similar to O-acetylserine sulfhydrylase, which generates L-cysteine using O-acetyl-L-serine with sulfide, and therefore, DcsD may be a synthase to generate O-ureido-L-serine using O-acetyl-L-serine and hydroxyurea. DcsG, which exhibits similarity to a family of enzymes with an ATP-grasp fold, may be an ATP-dependent synthetase converting O-ureido-D-serine into D-cycloserine. In the present study, to characterize the enzymatic functions of DcsC, DcsD, and DcsG, each protein was overexpressed in Escherichia coli and purified to near homogeneity. The biochemical function of each of the reactions catalyzed by these three proteins was verified by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and, in some cases, mass spectrometry. The results from this study demonstrate that by using a mixture of the three purified enzymes and the two commercially available substrates O-acetyl-L-serine and hydroxyurea, synthesis of D-cycloserine was successfully attained. These in vitro studies yield the conclusion that DcsD and DcsG are necessary for the syntheses of O-ureido-L-serine and D-cycloserine, respectively. DcsD was also able to catalyze the synthesis of L-cysteine when sulfide was added instead of hydroxyurea. Furthermore, the present study shows that DcsG can also form other cyclic d-amino acid analogs, such as D-homocysteine thiolactone.