Enzymatic synthesis of uridine diphosphate N-acetyl-D-mannosaminuronic acid.

Kawamura, Takashi; Ishimoto, Nobutoshi; Ito, Eiji

doi:10.1016/s0021-9258(19)86913-2

Cited by 53 publications

(30 citation statements)

References 30 publications

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“…Treatment of UDP-GlcNAc with the epimerase led to the generation of a new peak that was assigned to the product UDP-ManNAc (see Figure , traces A and B). Integration of the peaks gave a 10:1 ratio of UDP-GlcNAc:UDP-ManNAc, consistent with the reported equilibrium constant .…”

Section: Resultssupporting

confidence: 86%

“…The enzyme UDP-ManNAc dehydrogenase catalyzes the effectively irreversible 2-fold oxidation of UDP-ManNAc to UDP-ManNAcUA with the production of 2 equiv of NADH (Scheme ) . We have overexpressed this enzyme in E. coli 48 and used it to develop a continuous spectrophotometric assay of UDP-GlcNAc 2-epimerase activity.…”

Section: Resultsmentioning

confidence: 99%

“…Reduction of the ketone in the opposite stereochemical sense produces UDP-glucose and completes the epimerization process. Another example of an epimerase that acts at an “unactivated center” is UDP- N -acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase), which catalyzes the interconversion of UDP- N -acetylglucosamine (UDP-GlcNAc) and UDP- N -acetylmannosamine (UDP-ManNAc) (Scheme ). − Very little is known about the mechanism of this process or how the active site is able to accommodate two epimers that differ by the position of a relatively bulky acetamido group. In this paper we report studies that indicate this enzyme uses an unusual β-elimination/readdition strategy to carry out the inversion process …”

Section: Introductionmentioning

confidence: 99%

“…The E. coli UDP-GlcNAc 2-epimerase has been purified and was reported to be a dimer with a subunit molecular weight of about 38 000 Da. , Its activity was unaffected by the addition of either NAD + or NADP + indicating that if one of these cofactors were required it must be bound tightly enough to survive the purification procedure. Changes in the rate of epimerization as a function of UDP-GlcNAc concentration showed allosteric behavior with a Hill coefficient of 2.0.…”

Section: Introductionmentioning

confidence: 99%

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Eliminations in the Reactions Catalyzed by UDP-N-Acetylglucosamine 2-Epimerase

1997

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Mechanistic studies have been carried out on the bacterial enzyme UDP-N-acetylglucosamine 2-epimerase, which catalyzes the interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc). This enzyme is interesting because it epimerizes a stereocenter that does not bear an acidic proton, and therefore it cannot utilize a simple deprotonation/reprotonation mechanism. A coupled enzyme assay employing UDP-ManNAc dehydrogenase has been developed. The epimerization in D2O is found to be accompanied by the incorporation of deuterium into the C-2‘‘ position of both epimers, supporting a mechanism that ultimately involves a proton transfer at this position. The epimerization of [2‘‘-2H]UDP-GlcNAc is slowed by a primary kinetic isotope effect indicating that C−H bond cleavage is occurring during a rate-determining step of the reaction. A positional isotope exchange (PIX) experiment shows that an 18O label in the sugar-UDP bridging position will scramble into nonbridging diphosphate positions during enzymatic epimerization. These observations are consistent with a mechanism that proceeds via cleavage of the anomeric C−O bond, with 2-acetamidoglucal and UDP as enzyme-bound intermediates. Additional evidence for this mechanism is found in the unusual observation that during extended incubations, the intermediates are gradually released from the enzyme and accumulate in solution. These intermediates are formed by an anti elimination of UDP from UDP-GlcNAc and a syn elimination of UDP from UDP-ManNAc. It is likely that E1-like eliminations via oxocarbenium intermediates are involved in the reaction. Further experiments show that 3‘‘-deoxy-UDP-GlcNAc is not a substrate for the enzyme and that the enzyme does not contain a tightly bound NAD+ cofactor.

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Section: Resultssupporting

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Eliminations in the Reactions Catalyzed by UDP-N-Acetylglucosamine 2-Epimerase

1997

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“…wecB (a UDP- N -acetylglucosamine 2-epimerase) reversibly epimerizes at carbon position 2 between UDP-GlcNAc and UDP- N -acetylmannosamine (Sala et al, 1996 ; Morgan et al, 1997 ). WecC oxidizes UDP- N -acetylmannosamine in the presence of NAD+ to form UDP- N -acetyl-D-mannosaminuronic acid (ManNAcA) (Kawamura et al, 1979 ). The UDP-ManNAcA is the substrate to attach ManNAcA to the lipid IECA carried out by WecG (Barr and Rick, 1987 ; Barr et al, 1988 ).…”

Section: Introductionmentioning

confidence: 99%

wecB Gene of Salmonella Gallinarum Plays a Critical Role in Systemic Infection of Fowl Typhoid

et al. 2022

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Salmonella enterica serovar Gallinarum (S. Gallinarum) is a host-specific pathogen causing fowl typhoid, a severe systemic infection in poultry, which leads to substantial economic losses due to high morbidity and mortality in many developing countries. However, less is known about the pathogenic characteristics and mechanism of S. Gallinarum-induced systemic infection in chickens. In this study, we deleted the S. Gallinarum UDP-N-acetylglucosamine-1-phosphate transferase gene, which contributes to the biosynthesis of enterobacterial common antigen (ECA), and studied the pathogenicity of this wecB::Cm strain in a chicken model of systemic infection. The wecB::Cm mutant strain showed comparable growth but lower resistance to bile acid and nalidixic acid than the wild-type strain in vitro. In the oral infection model of chickens, the virulence of the wecB::Cm strain was significantly attenuated in vivo. Chickens infected with wild-type strain showed typical clinical signs and pathological changes of fowl typhoid and died between 6 and 9 days post-infection, and the bacteria rapidly disseminated to systemic organs and increased in the livers and spleens. In contrast, the wecB::Cm mutant strain did not cause chicken death, there were no significant clinical changes, and the bacterial numbers in the liver and spleen of the chickens were significantly lower than those of the chickens infected with the wild-type strain. In addition, the expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and CXCLi1 in the livers of wecB::Cm-infected chickens was significantly lower than that of the chickens infected with the wild-type strain. Furthermore, the attenuated wecB::Cm strain could persistently colonize the liver and spleen at low levels for up to 25 days post-infection and could induce a protective immune response in the chickens. These results indicate that the wecB gene is an important virulence factor of S. Gallinarum in the chicken model of systemic infection, and the avirulent wecB::Cm mutant could possibly be used as a live-attenuated vaccine strain for controlling fowl typhoid.

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Multiple Hydrogen Transfers in Enzyme Action

Spies

Toney

2006

Hydrogen‐Transfer Reactions

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Enzymatic synthesis of uridine diphosphate N-acetyl-D-mannosaminuronic acid.

Cited by 53 publications

References 30 publications

Eliminations in the Reactions Catalyzed by UDP-N-Acetylglucosamine 2-Epimerase

Eliminations in the Reactions Catalyzed by UDP-N-Acetylglucosamine 2-Epimerase

wecB Gene of Salmonella Gallinarum Plays a Critical Role in Systemic Infection of Fowl Typhoid

Multiple Hydrogen Transfers in Enzyme Action

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