Taxonomic distribution of proteins containing UDPG_MGDP_dh_C domains

The tree below includes only several representative species and genera. The complete taxonomic breakdown of all proteins containing UDPG_MGDP_dh_C domains can be accessed here. Click the counts or percentage values to display the corresponding proteins.

Properties and kinetic analysis of UDP-glucose dehydrogenase from group Astreptococci. Irreversible inhibition by UDP-chloroacetol.

Campbell RE, Sala RF, van de Rijn I, Tanner ME
J Biol Chem. 1997; 272: 3416-22

UDP-glucuronic acid is used by many pathogenic bacteria in theconstruction of an antiphagocytic capsule that is required for virulence.The enzyme UDP-glucose dehydrogenase catalyzes the NAD+-dependent 2-foldoxidation of UDP-glucose and provides a source of the acid. In the presentstudy the recombinant dehydrogenase from group A streptococci has beenpurified and found to be active as a monomer. The enzyme contains nochromophoric cofactors, and its activity is unaffected by the presence ofEDTA or carbonyl-trapping reagents. Initial velocity and productinhibition kinetic patterns are consistent with a bi-uni-uni-bi ping-pongmechanism in which UDP-glucose is bound first and UDP-glucuronate isreleased last. UDP-xylose was found to be a competitive inhibitor (Ki, 2.7microM) of the enzyme. The enzyme is irreversibly inactivated by uridine5'-diphosphate-chloroacetol due to the alkylation of an active sitecysteine thiol. The apparent second order rate constant for the inhibition(ki/Ki) was found to be 2 x 10(3) mM-1 min-1. Incubation with thetruncated compound, chloroacetol phosphate, resulted in no detectableinactivation when tested under comparable conditions. This supports thenotion that uridine 5'-diphosphate-chloroacetol is bound in the place ofUDP-glucose and is not simply acting as a nonspecific alkylating agent.

Purification and characterization of guanosine diphospho-D-mannosedehydrogenase. A key enzyme in the biosynthesis of alginate by Pseudomonasaeruginosa.

Roychoudhury S, May TB, Gill JF, Singh SK, Feingold DS, Chakrabarty AM
J Biol Chem. 1989; 264: 9380-5

Alginate-producing Pseudomonas aeruginosa are usually associated with thecystic fibrosis lung environment and contribute to the high mortalityrates observed among these patients. The present paper describes thepurification and enzymatic properties of guanosine diphospho-D-mannosedehydrogenase (EC 1.1.1.132), a key enzyme in alginate biosynthesis bymucoid P. aeruginosa. The enzyme was overproduced using a plasmid vectorcontaining algD (the gene encoding this enzyme) under control of the tacpromoter. It was purified from cell-free lysates by lowering the pH to5.0, heating the extract to 57.5 degrees C for 10 min, and discarding theprotein pellet. The enzyme was selectively precipitated from thesupernatant fraction with 45% acetone, resuspended in a 100 mMtriethanolamine acetate buffer, pH 7.6, and ultimately purified by Bio-SilTSK-400 gel filtration chromatography. The subunit molecular weight (Mr48,000) as well as the N-terminal amino acid sequence corresponded tothose predicted from the DNA sequence of algD. The native protein migratedas a hexamer of 290,000 molecular weight upon Bio-Gel A-1.5m gelfiltration chromatography. Kinetic analysis demonstrated an apparent Km of14.9 microM for the substrate GDP-D-mannose and 185 microM for thecofactor NAD+. GDP-D-mannuronic acid was identified as the enzyme reactionproduct. Several compounds (including GMP, ATP, GDP-D-glucose, andmaltose) were found to inhibit enzymatic activity. GMP, the most potent ofthese inhibitors, exhibited competitive inhibition with an apparent Ki of22.7 microM. Enzyme activity was also sensitive to the sulfhydryl groupmodifying agents iodoacetamide and p-hydroxymercuribenzoate. The additionof excess dithiothreitol restored enzyme activity, suggesting a possibleinvolvement of cysteine residues in enzymatic activity.