Establishment of anIn Vitrod-Cycloserine-Synthesizing System by UsingO-Ureido-l-Serine Synthase andd-Cycloserine Synthetase Found in the Biosynthetic Pathway

Abstract

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.

Coupled carbon, sulfur and nitrogen cycles of mixotrophic growth of Pseudomonas sp. C27 under denitrifying sulfide removal conditions

“…Apart from being oxidized to S 0 , sulfide is also involved in carbon metabolism. Sulfide was reported to react with O-Acetyl-L-serine to form L-cysteine and acetate by cysteine synthase A catalyzing (Kredich and Tomkins, 1996) by Lactobacillus (Bogicevic et al, 2012) or by Escherichia (Uda et al, 2013). Cysteine synthase A was claimed not only catalyzing synthesis of cysteine, but also having activity of desulfurization (Awano et al, 2005;Flint et al, 1996).…”

High-Level Heterologous Production of d -Cycloserine by Escherichia coli

“…6), suggesting that the added substrates are not fully used for the synthesis of D-CS. Because the D-CS production by the mutant E. coli BL21(DE3) strain increased, as described below, the saturation of the antibiotic production may be due to the limitation of OAS rather than to the depletion of ATP, which is necessary for the catalytic activity of DcsG (14).…”

“…Moreover, OAS is consumed by CysK, an OAS sulfhydrylase that forms L-cysteine from OAS and H 2 S as substrates. In addition, DcsD catalyzes the same reaction as does CysK, and the OAS sulfhydrylase activity of DcsD is superior to the enzymatic ability to synthesize O-ureido-L-serine (14). One of the strategies for increasing OAS, which is necessary for D-CS synthesis, is to reduce the above reactions.…”

“…L-Serine is O-acetylated by DcsE to generate O-acetyl-L-serine (OAS) (10, 13). The resultant OAS reacts with hydroxyurea (HU) to yield O-ureido-L-serine by use of DcsD, which is a pyridoxal phosphate-dependent enzyme (14). O-Ureido-Lserine is racemized by DcsC (14, 15), followed by cyclization with DcsG, which is a member of the ATP-grasp fold family of proteins (10, 14) (Fig.…”

2-Cyanoethyl-isoxazolin-5-one Is a Major Low Molecular Weight Nitrogenous Compound in Sweet Pea (<i>Lathyrus odoratus</i> L.)

“…Biosynthetic pathways of some isoxazoline compounds in Streptomyces have been studied. Ornithine is a candidate as a precursor in Streptomyces sviceus (Gould and Ju, 1992), whereas arginine and serine have been proposed to be precursors in S. lavendulae (Uda et al, 2013). However, basic biosynthetic pathways of 2-CEIX seems different even if amino acids or polyamines are prime precursors because of difference in their ling part structures.…”