[43] UDP-N-acetyl-d-mannosamine dehydrogenase from Escherichia coli

Kawamura, Takashi; Ishimoto, Nobutoshi; Ito, Eiji

doi:10.1016/0076-6879(82)83045-0

Cited by 8 publications

(13 citation statements)

References 2 publications

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“…However, the presence of Mg 2ϩ or Mn 2ϩ showed enhanced effect on the enzyme activity of WbpO Rf . This is similar to the effect of K ϩ on the enzymatic reaction of UDP-ManNAc dehydrogenase (60).…”

Section: Discussionsupporting

confidence: 66%

WbpO, a UDP-N-acetyl-d-galactosamine Dehydrogenase from Pseudomonas aeruginosa Serotype O6

Zhao¹,

Creuzenet²,

Bélanger³

et al. 2000

Journal of Biological Chemistry

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WbpO is associated with B-band lipopolysaccharide biosynthesis in Pseudomonas aeruginosa serotype O6. This protein is thought to catalyze the enzymatic conversion of UDP-N-acetyl-D-galactosamine (UDP-GalNAc) to UDP-N-acetyl-D-galactosaminuronic acid (UDP-GalNAcA). WbpO was overexpressed with a C-terminal hexahistidine tag. The soluble form of expressed WbpO (WbpO Sol ) exhibited a secondary structure with 29.2% ␣-helix and 20.1% ␤-strand. However, no enzymatic activity could be detected using either high performance anion exchange chromatography or capillary electrophoresis-mass spectrometry analysis. An insoluble form of expressed WbpO was purified in the presence of guanidine hydrochloride by immobilized metal ion affinity chromatography. After refolding, this preparation of WbpO (designated as WbpO Rf ) exhibited stable secondary structure at pH 7.5 to 8.2, and it was enzymatically active. Capillary electrophoresis-mass spectrometry and tandem mass spectrometry analysis showed that WbpO Rf catalyzed the conversion of UDP-GalNAc to UDP-GalNAcA. 26 and 22% of the substrate could be converted to UDP-GalNAcA in the presence of NAD ؉ and NADP ؉ as the cofactors, respectively. The K m values of WbpO Rf for UDP-GalNAc, NAD ؉ , and NADP ؉ were 7.79, 0.65, and 0.44 mM, respectively. WbpO Rf can also catalyze the conversion of UDP-GlcNAc to UDP-GlcNAcA. In conclusion, this is the first report of the overexpression, purification, and biochemical characterization of an NAD ؉ /NADP ؉ -dependent UDP-GalNAc dehydrogenase. Our results also complete the biosynthetic pathway for GalNAcA that is part of the O-antigen of P. aeruginosa serotype O6 lipopolysaccharide.

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Section: Discussionsupporting

confidence: 66%

WbpO, a UDP-N-acetyl-d-galactosamine Dehydrogenase from Pseudomonas aeruginosa Serotype O6

Zhao¹,

Creuzenet²,

Bélanger³

et al. 2000

Journal of Biological Chemistry

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“…18,33 A direct assay using ion-paired reversed-phase high-performance liquid chromatography (HPLC) was also reported. 18 We developed an effective direct capillary electrophoresis approach to allow the separation and detection of UDP-GlcNAc, UDP-ManNAc, 2-acetamidoglucal, and UDP (Figs.…”

Section: Resultsmentioning

confidence: 99%

“…Bacterial non-hydrolyzing UDP-GlcNAc 2-epimerases from Bacillus cereus 43 and E. coli O14 K7 H 18,33 were shown to be allosteric enzymes and UDP-GlcNAc was absolutely required for their UDP-ManNAc 2-epimerization catalytic activity. 33 In our study, when chemoenzymatically synthesized pure UDP-ManNAc was used as the only starting material, NmSacA-His 6 successfully catalyzed the 2-epimerase reaction for the formation of UDP-GlcNAc.…”

Section: Resultsmentioning

confidence: 99%

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Characterizing non-hydrolyzing Neisseria meningitidis serogroup A UDP-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase using UDP-N-acetylmannosamine (UDP-ManNAc) and derivatives

Zhang

Muthana

et al. 2016

Carbohydrate Research

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Neisseria meningitidis serogroup A non-hydrolyzing uridine 5′-diphosphate-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase (NmSacA) catalyzes the interconversion between UDP-GlcNAc and uridine 5′-diphosphate-N-acetylmannosamine (UDP-ManNAc). It is a key enzyme involved in the biosynthesis of the capsular polysaccharide [-6ManNAcα1-phosphate-]n of N. meningitidis serogroup A, one of the six serogroups (A, B, C, W-135, X, and Y) that account for most cases of N. meningitidis-caused bacterial septicemia and meningitis. N. meningitidis serogroup A is responsible for large epidemics in the developing world, especially in Africa. Here we report that UDP-ManNAc could be used as a substrate for C-terminal His6-tagged recombinant NmSacA (NmSacA-His6) in the absence of UDP-GlcNAc. NmSacA-His6 was activated by UDP-GlcNAc and inhibited by 2-acetamidoglucal and UDP. Substrate specificity study showed that NmSacA-His6 could tolerate several chemoenzymatically synthesized UDP-ManNAc derivatives as substrates although its activity was much lower than non-modified UDP-ManNAc. Homology modeling and molecular docking revealed likely structural determinants of NmSacA substrate specificity. This is the first detailed study of N. meningitidis serogroup A UDP-GlcNAc 2-epimerase.

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“…Initial studies of UDP-D-GlcNAc C-2 epimerase from E. coli proposed that epimerization occurs via a ketone at the C-3 position (32). However, recent work on RffE led to the identification of a novel mechanism involving the anti elimination and further syn addition of the O-UDP moiety to the anomeric carbon (26,(33)(34)(35)(36). This mechanism has also been demonstrated in a hydrolyzing UDP-D-GlcNAc C-2 epimerase, NeuC, from Neisseria meningitidis (37,38).…”

Section: Table I Nmr Analysis Of Compounds Udp-␤-l-pnenac and Udp-␤-lmentioning

confidence: 99%

Biosynthesis of UDP-N-acetyl-l-fucosamine, a Precursor to the Biosynthesis of Lipopolysaccharide in Pseudomonas aeruginosa Serotype O11

Mulrooney

Poon

McNally

et al. 2005

Journal of Biological Chemistry

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UDP-N-acetyl-L-fucosamine is a precursor to L-fucoThose results led us to propose a pathway for the biosynthesis of UDP-L-FucNAc; however, the exact enzymatic activity of each of these proteins has not been defined. Here, we describe a fast protein liquid chromatography (FPLC)-based anion-exchange procedure, which allowed the separation and purification of the products of C-2 epimerization due to WbjD. Also, the application of a cryogenically cooled probe in NMR spectrometry offers the greatest sensitivity for determining the structures of minute quantities of materials, allowing the identification of the final product of the pathway. Our results showed that WbjB is bifunctional, catalyzing firstly C-4, C-6 dehydration and secondly C-5 epimerization in the reaction with the substrate UDP-D-GlcNAc, producing two intermediates. WbjC is also bifunctional, catalyzing C-3 epimerization of the second intermediate followed by reduction at C-4. The FPLC-based procedure provided good resolution of the final product of WbjD reaction from its epimer/substrate UDP-L-PneNAc, and the use of the cryogenically cooled probe in NMR revealed unequivocally that the final product is UDP-L-FucNAc.

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[43] UDP-N-acetyl-d-mannosamine dehydrogenase from Escherichia coli

Cited by 8 publications

References 2 publications

WbpO, a UDP-N-acetyl-d-galactosamine Dehydrogenase from Pseudomonas aeruginosa Serotype O6

WbpO, a UDP-N-acetyl-d-galactosamine Dehydrogenase from Pseudomonas aeruginosa Serotype O6

Characterizing non-hydrolyzing Neisseria meningitidis serogroup A UDP-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase using UDP-N-acetylmannosamine (UDP-ManNAc) and derivatives

Biosynthesis of UDP-N-acetyl-l-fucosamine, a Precursor to the Biosynthesis of Lipopolysaccharide in Pseudomonas aeruginosa Serotype O11

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