Biosynthesis of UDP-glucuronic acid and UDP-galacturonic acid in Bacillus cereus subsp. cytotoxis NVH 391-98.
The food borne pathogen Bacillus cereus produces uronic acid-containing glycans that are secreted in a shielding biofilm environment, and certain alkaliphilic Bacillus deposit uronate-glycan polymers in the cell wall when adapting to alkaline environments. The source of these acidic sugars is unknown and, in the present study, we describe the functional identification of an operon in Bacillus cerues subsp. cytotoxis NVH 391-98 that comprises genes involved in the synthesis of UDP-uronic acids in Bacillus spp. Within the operon, a UDP-glucose 6-dehydrogenase converts UDP-glucose in the presence of NAD(+) to UDP-glucuronic acid and NADH, and a UDP-GlcA 4-epimerase (UGlcAE) converts UDP-glucuronic acid to UDP-galacturonic acid. Interestingly, in vitro, both enzymes can utilize the TDP-sugar forms as well, albeit at lower catalytic efficiency. Unlike most of the very few bacterial 4-epimerases that have been characterized, which are promiscuous, the B. cereus UGlcAE enzyme is very specific and cannot use UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylglucosaminuronic acid or UDP-xylose as substrates. Size exclusion chromatography suggests that UGlcAE is active as a monomer, unlike the dimeric form of plant enzymes; the Bacillus UDP-glucose 6-dehydrogenase is also found as a monomer. Phylogenic analysis further suggests that the Bacillus UGlcAE may have evolved separately from other bacterial and plant epimerases. Our results provide insight into the formation and function of uronic acid-containing glycans in the lifecycle of B. cereus and related species containing homologous operons, as well as a basis for determining the importance of these acidic glycans. We also discuss the ability to target UGlcAE as a drug candidate.
Complex Carbohydrate Research Center (CCRC), University of Georgia, Athens, GA 30602, USA.
Bryan
B
Broach
Xiaogang
X
Gu
Maor
M
Bar-Peled
D001409:Bacillus cereus / Q000235:genetics / Q000378:metabolism*; D002238:Carbohydrate Epimerases / Q000235:genetics* / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
D011994:Recombinant Proteins; D014539:Uridine Diphosphate Sugars; D014535:Uridine Diphosphate Glucuronic Acid; C050306:UDP-galacturonic acid; D002238:Carbohydrate Epimerases