Appearance of Uridine 5′‐Diphospho‐N‐Acetylglucosamine‐4‐Epimerase during Sporulation of Bacillus megaterium

Abstract

In a biosynthetic study of the spore coat of Bacillus megaterium ATCC 12872 spore with galactosamine phosphate as a major component of the outer coat, high-performance liquid chromatography (HPLC) and enzyme immunoassay were applied for the measurement of UDP-N-acetylglucosamine-4-epimerase [EC 5.1.3.7] activity and the enzyme protein concentration, respectively. The new HPLC system using an ion-pair (or anion-exchange) column allowed us to determine successfully the enzyme activity and its application, proving that the specific activity of the enzyme in the cells increased at the later stage of sporulation. This increase in activity was parallel to the induction of enzyme protein synthesis, which was detected by sandwich enzyme immunoassay using antiserum to the purified enzyme. These results suggested that the regulation of this enzyme is at the genetic level and it plays an important role in the outer coat synthesis in the later sporulation stage of B. megaterium.The spore of Bacillus megaterium ATCC 12872 has sugar components, composed almost entirely of polymerized galactosamine-6-phosphate, in the outer coat (9, 10), which has been called the "exosporium" by Gerhardt's group (7). One of the enzymes involved in the synthesis of galactosamine-6-phosphate polymer must be UDP-N-acetylglucosamine-4-epimerase [EC 5.1.3.7], which catalyzes the conversion of UDP-N-acetylglucosamine (UDP-GlcNAc) to UDP-N-acetylgalactosamine (UDPGalNAc). Because the outer coat is chemically and morphologically specific to spores and is synthesized at the later stage of spore formation, it is assumed that the biosynthesis of this structure is regulated during the cell cycle. As the first step in this direction, the activity and the amount of UDP-GlcNAc-4-epimerase had to be determined in sporulating cells.Previous assays for this enzyme were carried out by quantitative analysis of galactosamine or glucosamine by paper chromatography (2) and/or enzymatic analysis (6) after acid hydrolysis. Without acid hydrolysis, measurement of UDPGlcNAc or UDP-GalNAc produced was performed using UDP-glucose dehydrogenase (8) or lectin affinity chromatography (11). These methods were not sufficient for our experiments because of inaccuracy and the long time required by the complicated procedures involved.

New UDP-GlcNAc C4 Epimerase Involved in the Biosynthesis of 2-Acetamino-2-deoxy-l-altruronic Acid in the O-Antigen Repeating Units of Plesiomonas shigelloides O17

“…The activity was first described in crude extracts from Bacillus subtilis () and later reported or inferred in a variety of prokaryotic and eukaryotic species ( − ). The potential location of the gene encoding UDP-GlcNAc 4-epimerase activity has been reported by Estrela et al ().…”

UDP‐N‐Acetylglucosamine 4′‐Epimerase from the Intestinal Protozoan Giardia Intestinalis Lacks UDP‐Glucose 4′‐Epimerase Activity1

“…Epimerase activity has been detected in a number of mammals, such as humans and rats (Thoden et al 2001a; Wohlers et al 1999), and a number of bacteria, such as Bacillus (Nishikawa et al 1986), Pseudomonas aeruginosa strain PA01 (Creuzenet et al 2000), and Yersinia (Bengoechea et al 2002). The enzyme from these organisms is able to catalyze two similar, but distinct, reversible reactions: (1) UDP‐GlcNAc to UDP‐GalNAc and (2) UDP‐Glc to UDP‐Gal (EC 5.1.3.2).…”