Enzymatic Synthesis of Cytidine Diphosphate 3,6-Dideoxyhexoses

Rubenstein, P A; Strominger, Jack L.

doi:10.1016/s0021-9258(19)42542-8

Cited by 40 publications

(21 citation statements)

References 12 publications

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“…5 In the formation of the antigenic determinant deoxyhexoses the resulting CDP-4-keto-6-deoxyglucose is then further deoxygenated at C-3 by the action of a pyridoxamine phosphate (PMP) and iron-sulfur clustercontaining enzyme, E 1 , and an electron-transfer protein, E 3 , to give CDP-4-keto-3,6-dideoxyglucose. 3 This reaction has been studied extensively 6,7 and involves a novel PMP-mediated radical reaction. 8 In the antibiotic deoxy sugars a common modification following 6-deoxygenation is the loss of the oxygen function from C-2, but very little is known about this process or the enzyme(s) catalyzing it.…”

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confidence: 99%

Mechanism of the 2-Deoxygenation Step in the Biosynthesis of the Deoxyhexose Moieties of the Antibiotics Granaticin and Oleandomycin

1999

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Mechanism of the 2-Deoxygenation Step in the Biosynthesis of the Deoxyhexose Moieties of the Antibiotics Granaticin and Oleandomycin

1999

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“…gives rise to the actual deoxygenation product, CDP-4-keto-3,6-dideoxy-D-glucose (6), that had been isolated and identified (Rubenstein & Strominger, 1974b). Our recent characterization of the cloned Ei expressed in Escherichia coli has revealed, in addition to PMP, the presence of a [2Fe-2S] center whose EPR characteristics are akin to those of adrenodoxin and putidaredoxin (Thorson & Liu, 1993a).…”

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confidence: 99%

Mechanistic Studies on CDP-6-deoxy-L-threo-D-glycero-4-hexulose 3-Dehydrase: Identification of His-220 as the Active-Site Base by Chemical Modification and Site-Directed Mutagenesis

Lei

Ploux²,

Liu³

1995

Biochemistry

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CDP-6-deoxy-L-threo-D-glycero-4-hexulose 3-dehydrase (E1) purified from Yersinia pseudotuberculosis is a pyridoxamine 5'-phosphate (PMP) dependent iron-sulfur-containing enzyme which catalyzes the C-O bond cleavage at C-3 of its substrate leading to the formation of 3,6-dideoxyhexose. This enzyme is rapidly inactivated by diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees C. The inactivation of E1 by DEP, which is reversible upon treatment of hydroxylamine, appears to be attributable solely to the modification of histidine residues. The fact that coincubation of E1 with its substrate gave almost total protection against DEP inactivation and that only one less histidine residue was modified in the presence of substrate strongly suggested that inactivation is due to the modification of only one reactive histidine residue which resides in or near the active site of E1 and is critical for E1's activity. Sequence alignment between the translated ascC (E1) gene and several representative pyridoxal 5'-phosphate (PLP)/PMP dependent enzymes revealed that three of the four invariant residues, glycine, aspartate, and arginine found in all other aminotransferases, are conserved in the E1 sequence (G169, D191, and R403). However, the highly conserved lysine is replaced by a histidine residue (H220) in E1. In order to test whether H220 plays an essential role in E1 catalysis, H220N mutant was constructed and the encoding protein was found to exhibit nearly identical physical characteristics as the wild-type E1. Interestingly, the mutant protein had lost most of its catalytic activity, and one less histidine residue was modified upon treatment of H220N-mutated protein with DEP. Such a single-point mutation also impaired E1's capability of catalyzing the solvent hydrogen exchange at C-4' position of the PMP coenzyme. Our findings strongly suggested that H220 is most likely the active-site base which abstracts the C-4' proton from the PMP-substrate adduct and initiates the catalysis. Furthermore, E1's preservation of other invariant residues found in many PLP/PMP dependent enzymes allowed a speculation of their roles in E1 catalysis.(ABSTRACT TRUNCATED AT 400 WORDS)

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“…[4(F)-2H] NADPH or [4-(J? )-3H]NADPH was prepared from the appropriate isotopically substituted isocitrate with the F-specific enzyme isocitrate dehydrogenase, by a modification of the procedure described by Rubenstein & Strominger (1974). NADP"1", 60 µ , and D-iAreo-isocitrate, 90 µ (180 µ of dl-iAreo-isocitrate), were dissolved in 10 mL of glycylglycine buffer, pH 7.5, containing 1 mM MgCl2.…”

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Kinetic isotope effects on the oxidation of reduced nicotinamide adenine dinucleotide phosphate by the flavoprotein methylenetetrahydrofolate reductase

Vanoni¹,

Matthews²

1984

Biochemistry

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Previous work from this laboratory has established that the NADPH-menadione oxidoreductase reaction catalyzed by methylenetetrahydrofolate reductase from pig liver proceeds by Ping Pong Bi Bi kinetics and that the reductive half-reaction is rate limiting in steady-state turnover. We have now shown that methylenetetrahydrofolate reductase stereo-specifically removes the pro-S hydrogen from the 4-position of NADPH. During the oxidation of [4(S)-3H]NADPH, we observed a kinetic isotope on V/KNADPH of 10.8 +/- 0.4. When comparing the rates of oxidation of [4(S)-2H]NADPH and [4(S)-1H]NADPH, we measure kinetic isotope effects on V of 4.78 +/- 0.15 and on V/KNADPH of 4.54 +/- 0.59. When oxidation of [4(R)-2H]NADPH and [4(R)-1H]NADPH is compared, the secondary kinetic isotope effect on V is 1.04 +/- 0.01. When the NADPH-menadione oxidoreductase reaction is catalyzed in tritiated water, no incorporation of solvent tritium into residual NADPH is observed. We conclude from these observations that the oxidation of NADPH is largely or entirely rate limiting in the reductive half-reaction and, hence, in NADPH-menadione oxidoreductase turnover at saturating menadione concentration. In the presence of saturating NADPH, the flavin reduction proceeds with a rate constant of 160 S-1, which is at least 29-fold slower than estimates of the lower limit for the diffusion-limited rate constant characterizing NADPH binding to the enzyme under physiological conditions. Albery & Knowles have defined criteria for perfection in enzyme catalysis [Albery, W. J., & Knowles, J.R. (1976) Biochemistry 15, 5631-5640].(ABSTRACT TRUNCATED AT 250 WORDS)

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Enzymatic Synthesis of Cytidine Diphosphate 3,6-Dideoxyhexoses

Cited by 40 publications

References 12 publications

Mechanism of the 2-Deoxygenation Step in the Biosynthesis of the Deoxyhexose Moieties of the Antibiotics Granaticin and Oleandomycin

Mechanism of the 2-Deoxygenation Step in the Biosynthesis of the Deoxyhexose Moieties of the Antibiotics Granaticin and Oleandomycin

Mechanistic Studies on CDP-6-deoxy-L-threo-D-glycero-4-hexulose 3-Dehydrase: Identification of His-220 as the Active-Site Base by Chemical Modification and Site-Directed Mutagenesis

Kinetic isotope effects on the oxidation of reduced nicotinamide adenine dinucleotide phosphate by the flavoprotein methylenetetrahydrofolate reductase

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