l-Ribulose 5-Phosphate 4-Epimerase of Aerobacter aerogenes

Deupree, Jean D.; Wood, William F.

doi:10.1016/s0021-9258(18)62946-1

Cited by 24 publications

(41 citation statements)

References 24 publications

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“…34 Since the N-terminal portion of L-ribulose-5-phosphate 4-epimerase is homologous to a class II L-fuculose-1-phosphate aldolase from E. coli, this epimerase was thus investigated whether it makes use of an aldol chemistry. 35,7 Results from these and other studies 34,[36][37][38] suggest that the epimerase is capable of promoting carboncarbon bond cleavage and support a mechanism that follows a retroaldol cleavage process, generating glycolaldehyde phosphate and the metal-bound enolate of dihydroxyacetone. Rotation of the aldehyde carbonyl exposes the opposite face, and upon aldol addition of the enolate, the epimeric sugar is formed.…”

Section: Discussionmentioning

confidence: 74%

New Insights into the Mechanism of CDP-d-Tyvelose 2-Epimerase: An Enzyme-Catalyzing Epimerization at an Unactivated Stereocenter

2000

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Tyvelose is a 3,6-dideoxyhexose found in the O-antigen of Yersinia pseudotuberculosis IVA and is the only member of this class of sugars to be produced directly from another 3,6-dideoxyhexose, paratose. The C-2 epimerization required for this conversion has been proposed to be catalyzed by CDP-d-tyvelose 2-epimerase. This enzyme is intriguing since it belongs to a group of epimerases, including the well-studied UDP-d-galactose 4-epimerase, that can invert unactivated stereocenters. To study the mechanism of this enzyme, we have cloned and expressed the tyv gene that encodes CDP-d-tyvelose 2-epimerase. The purified tetrameric protein contains approximately one equivalent of bound NAD+ per monomer and a small fraction of NADH. Four possible mechanisms involving NAD+ can be proposed for this enzyme; two involve oxidation at C-2 of the substrate, while the other two require oxidation at C-4. In a previous contribution, we presented preliminary data that supported a retro-aldol-type mechanism initiated by C-4 oxidation. However, this mechanism was refuted by further investigations, which revealed that the 4-fluoro analogue of CDP-d-paratose could be turned over by the enzyme. More importantly, the direct transfer of a deuterium from C-2 of the labeled substrate to the enzyme-bound NAD+ was observed by mass spectrometry. These results suggest that epimerization is in fact initiated by oxidation at C-2, followed by the transfer of the hydride from the transiently formed NADH to the opposite side of the 2-hexulose intermediate.

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Section: Discussionmentioning

confidence: 74%

New Insights into the Mechanism of CDP-d-Tyvelose 2-Epimerase: An Enzyme-Catalyzing Epimerization at an Unactivated Stereocenter

2000

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“…The epimerization of the C-2' hydroxyl group of a purine nucleoside or nucleotide in ara-A biosynthesis is unique in that it is the first example of this type reaction that occurs at the level of a pentose in a nucleoside or nucleotide. The data available to date on the 2and 4-epimerizations show that these reactions occur as either the nucleoside diphosphate sugar (such as UDP-galactose 4'-epimerase and UDP-vVacetylglucosamine 2'-epimerase) or as the sugar phosphate (such as ribulose 5-phosphate 4-epimerase) (Nelsesteun and Kirkwood, 1970; Salo and Fletcher, 1970;Deupree and Wood, 1970)). In view of the data presented here on the epimerization of the C-2' hydroxyl of the ribose moiety of adenosine to form ara-A, it will be of extreme interest to elucidate the mechanism of the epimerization in this nucleoside or nucleotide interconversion.…”

Section: Discussionmentioning

confidence: 99%

Nucleoside antibiotics. Biosynthesis of arabonofuranosyladenine by Streptomyces antibioticus. 10

Farmer¹,

Suhadolnik²

1972

Biochemistry

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“…The epimerase serves to link the arabinose metabolic pathway to the pentose phosphate pathway and allows the bacteria to use arabinose as an energy source (1). The enzyme has a molecular mass of 102 kDa (2,3), and is composed of four identical 25.5 kDa subunits (4,5). The epimerase is also known to require one divalent metal cation per subunit for activity (6).…”

mentioning

confidence: 99%

“…Thus, the proton at the C-4 position of the epimeric sugars is not acidic, and a simple deprotonation/reprotonation mechanism can be ruled out. Furthermore, the enzyme has been shown not to use an NAD + cofactor during catalysis (2), and therefore does not employ an oxidation/reduction mechanism analogous to that of UDP-galactose 4-epimerase (9,10). Other studies have shown that the reaction proceeds without the incorporation of solvent-derived oxygen or hydrogen atoms and that there is no primary kinetic isotope effect in the epimerization of either D-[4-3 H]Xu5P or L-[4-2 H]Ru5P (11)(12)(13).…”

mentioning

confidence: 99%

Epimerization via Carbon−Carbon Bond Cleavage. l-Ribulose-5-phosphate 4-Epimerase as a Masked Class II Aldolase

Johnson

Tanner

1998

Biochemistry

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Studies indicating that the E. coli L-ribulose-5-phosphate 4-epimerase employs an "aldolase-like" mechanism are reported. This NAD+-independent enzyme epimerizes a stereocenter that does not bear an acidic proton and therefore it cannot utilize a simple deprotonation-reprotonation mechanism. Sequence similarities between the epimerase and the class II l-fuculose-1-phosphate aldolase suggest that the two may be evolutionarily related and that the epimerization may occur via carbon-carbon bond cleavage and re-formation. Conserved residues thought to provide the metal ion ligands of the epimerase have been modified using site-directed mutagenesis. The resulting mutants show low kcat values in addition to a reduced affinity for Zn2+. These observations serve to establish that there is a structural link between between the active site geometry of the epimerase and the aldolase. In addition, the H97N mutant was found to catalyze the condensation of dihydroxyacetone and glycolaldehyde phosphate to produce a mixture of L-ribulose-5-phosphate and D-xylulose-5-phosphate. This observation of aldolase activity establishes that the epimerase active site is capable of promoting carbon-carbon bond cleavage. Furthermore, glycolaldehyde phosphate was shown to be a competitive inhibitor of the mutant enzyme (KI = 0.37 mM) but not of the wild-type enzyme. The mutation apparently causes the epimerase to become "leaky" and enables it to bind/generate the normal reaction intermediates from the unbound aldol cleavage products.

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l-Ribulose 5-Phosphate 4-Epimerase of Aerobacter aerogenes

Cited by 24 publications

References 24 publications

New Insights into the Mechanism of CDP-d-Tyvelose 2-Epimerase: An Enzyme-Catalyzing Epimerization at an Unactivated Stereocenter

New Insights into the Mechanism of CDP-d-Tyvelose 2-Epimerase: An Enzyme-Catalyzing Epimerization at an Unactivated Stereocenter

Nucleoside antibiotics. Biosynthesis of arabonofuranosyladenine by Streptomyces antibioticus. 10

Epimerization via Carbon−Carbon Bond Cleavage. l-Ribulose-5-phosphate 4-Epimerase as a Masked Class II Aldolase

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