Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

CDP-6-deoxy-delta 3,4-glucoseen reductase (E3), which catalyzes the reduction of the C-3 deoxygenation step during the formation of CDP-ascarylose, a 3,6-dideoxyhexose found in the lipopolysaccharide of Yersinia pseudotuberculosis, has been expressed at high level in Escherichia coli (670 times over the wild-type strain). This flavoenzyme, which also contains one plant ferredoxin type [2Fe-2S] cluster, was inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide. In both cases the inactivation followed a pseudo first order kinetics. The second order rate constant for the reaction of DTNB with E3 was 0.25 mM-1 min-1 at 20 degrees C, pH 8.0. Detailed characterization of the inactivated enzyme showed that neither the flavin nor the [2Fe-2S] cluster was altered during inactivation. Since this inactivation was reversible by treating the inactivated enzyme with 1 mM D,L-dithiothreitol (DTT), it was concluded that only cysteine residues were modified during inactivation. Analysis of the inactivation using the method developed by Tsou revealed that two cysteines react with DTNB at similar rates and modification of either one is enough to impair E3's activity. Tryptic digestion of E3 labeled with N-ethyl[2,3-14C]maleimide, followed by fractionation of the digest by high performance liquid chromatography, gave two labeled peptides, both of which were separately isolated as a pair of interconvertible diastereoisomers. Sequence analysis of these labeled peptides allowed the identification of Cys-75 and Cys-296 as the reactive cysteine residues. Interestingly, the C75S and C296S mutant proteins exhibit identical physical and comparable catalytic properties as the wild-type enzyme. Since Cys-296 is a conserved residue in the NAD(P) binding domain of enzymes belonging to the same class, this residue may be involved in stabilizing the charge-transfer complex between E3 and NADH, thus facilitating hydride transfer from the nicotinamide nucleotide to flavin. A chemically modified Cys-75 which is immediately adjacent to the [2Fe-2S] center in E3 may prevent the proper juxtaposition of the redox centers and thus impede electron transfer leading to enzyme inactivation. These results may be useful for placing constraints on the peptide folding comprising the active site of E3 for electron transfer between NADH, FAD, and the [2Fe-2S] center.
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PMID:Mechanistic studies on CDP-6-deoxy-delta 3,4-glucoseen reductase: the role of cysteine residues in catalysis as probed by chemical modification and site-directed mutagenesis. 770 27

The CDP-6-deoxy-delta 3,4-glucoseen reductase (E3) is a NADH-dependent enzyme which catalyzes the key reduction of the C-3 deoxygenation step during the formation of CDP-ascarylose, a 3,6-dideoxyhexose found in the lipopolysaccharide of Yersinia pseudotuberculosis. This highly purified enzyme is also a NADH oxidase capable of mediating the direct electron transfer from NADH to O2, forming H2O2. While previous work showed that E3 contains no common cofactor, one FAD and one plant ferredoxin type [2Fe-2S] center were found in this study to be associated with each molecule of E3. The iron-sulfur center is essential for E3 activity since bleaching of the [2Fe-2S] center leads to inactive enzyme. These results suggest that E3 employs a short electron-transport chain composed of both FAD and the iron-sulfur center to shuttle electrons from NADH to its acceptor. The order of electron flow, as indicated by EPR measurement with partially reduced E3, starts with hydride reduction of FAD by NADH. The iron-sulfur cluster, receiving electrons one at a time from the reduced flavin, relays the reducing equivalents via another iron-sulfur center in the active site of E1 to its final acceptor, the E1-bound PMP-glucoseen adduct. The participation of a one-electron-carrying iron-sulfur center in this reduction is advantageous since both electrons are dispatched from the same redox state of the prosthetic group, allowing electrons of equal energy to be delivered to the final acceptor.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cofactor characterization and mechanistic studies of CDP-6-deoxy-delta 3,4-glucoseen reductase: exploration into a novel enzymatic C-O bond cleavage event. 821 67

A mercuric ion-reducing flavoprotein was purified from Yersinia enterocolitica 138A14 using dye matrix affinity chromatography. The purified enzyme had a characteristic absorption spectrum similar to those of flavin compounds, and FAD was detected as a part of the purified enzyme by thin-layer chromatography. Freshly purified preparations of the enzyme showed a single band on SDS polyacrylamide gel electrophoresis with a molecular weight of 70,000. The isolated enzyme had a molecular weight of about 200,000 as determined by gel filtration and disc gel electrophoresis. These results suggest an apparently trimeric structure of the enzyme. Dithiothreitol treatment disrupted the trimer into a dimeric structure of 140,000. Along with ageing, as well as limited proteolytic digestion, the enzyme evolved to give a dimeric molecule of 105,000 composed of two identical subunits of 52,000. The combination of the purified enzyme with HgCl2, or unexpectedly with merthiolate, oxidised the NADPH, which was followed spectrophotometrically. The Km for HgCl2 was dependent on the concentration of exogenous thiol compounds. A comparison of physical properties as well as kinetic characteristics indicated that the enzyme from Y. enterocolitica 138A14 is similar to mercuric reductases isolated from other mercury-resistant bacteria.
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PMID:Purification and properties of mercuric reductase from Yersinia enterocolitica 138A14. 846 20

The conversion of CDP-4-keto-6-deoxy-D-glucose to CDP-4-keto-3,6-dideoxy-D-glucose is a key step in biosynthesis of ascarylose, the terminal dideoxyhexose of the O-antigen tetrasaccharide of the lipopolysaccharide from Yersinia pseudotuberculosis V. This transformation is catalyzed by two enzymes: CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), which contains a pyridoxamine and a [2Fe-2S] center, and an NADH-dependent CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3), which contains both an FAD and a [2Fe-2S] center. E1 reacts to form a Schiff base with CDP-4-keto-6-deoxy-D-glucose and catalyzes the elimination of the hydroxyl at position 3 of the glucose moiety, resulting in the formation of a covalently bound CDP-6-deoxy-delta(3,4)-glucoseen intermediate. E3 transfers electrons from NADH to E1, which uses these to reduce the delta(3,4)-glucoseen bond to produce CDP-4-keto-3,6-dideoxy-D-glucose. In this work, we have investigated the reductive half-reaction of E3 using both single wavelength and diode array stopped flow absorbance spectroscopy. We find that NADH binds to both oxidized (Kd = 52.5 +/- 2 microM) and two-electron-reduced (Kd = 12.1 +/- 1 microM) forms of E3. Hydride transfer from NADH to the FAD moiety occurs at 107.5 +/- 3 s-1 and exhibits a 10-fold deuterium isotope effect when (4R)-[2H]NADH is substituted for NADH. Following the hydride transfer reaction, NAD+ is released at 42.5 +/- 1 s-1 and electron transfer from the reduced FAD to the [2Fe-2S] center occurs rapidly. The extent of the intramolecular electron transfer reaction is pH-dependent with a pKa of 7.3 +/- 0.1, which may represent the ionization state of the N-1 position of the FAD hydroquinone of E3. Finally, E3 is converted to the three-electron-reduced state in a slow disproportionation reaction that consumes NADH: The [2Fe-2S] center of E3 was selectively disassembled by titration with mersalyl to give E3(apoFeS). The properties of this form of the enzyme are compared to those of the holoenzyme. Similarities and differences of the reductive half-reactions of E3 and related iron-sulfur flavoenzymes are discussed.
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PMID:Kinetics of the reductive half-reaction of the iron-sulfur flavoenzyme CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase. 867 75

Studies of the biosynthesis of ascarylose, a 3,6-dideoxyhexose found in the lipopolysaccharide of Yersinia pseudotuberculosis V, have shown that the C-3 deoxygenation is a process consisting of two enzymatic steps. The first enzyme involved in this transformation is CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), which is a pyridoxamine 5'-phosphate dependent iron-sulfur protein. The second catalyst, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase, formally called CDP-6-deoxy-delta(3,4)-glucoseen reductase (E3), is an NADH dependent plant type [2Fe-2S] containing flavoenzyme. To better understand the electron transfer carried out by these two enzymes, the potentials of the E1 and E3 redox cofactors were determined spectroelectrochemically. At pH 7.5, the midpoint potential of the E3 FAD was found to be -212 mV, with the FADox/FADsq couple (E1o') and the FADsq/FADhq couple (E2o') calculated to be -231 and -192 mV, respectively. However, the E1o' and E2o' of the FAD in E3(apoFeS) at pH 7.5 were estimated to be -215 and -240 mV, respectively, which are quite different from those of the holo-E3, suggesting a significant effect of the iron-sulfur center on the redox properties of the flavin coenzyme. Our data also showed that the midpoint potential of the E3 iron-sulfur is -257 mV and that of the E1 [2Fe-2S] center is -209 mV. These values indicated a thermodynamic barrier to the proposed electron transfer of NADH->FAD=>E3[2Fe-2S]->E1[2Fe-2S] at pH 7.5. Regulation of electron transfer by several mechanisms is possible and experiments were performed to examine ways of overcoming the unfavorable electron transfer energetics in the E1/E3 system. It was found that both binding of E3 with NAD+ and complex formation between E3 and E1 showed no effect on the midpoint potentials of the E3 FAD and iron-sulfur center. Interestingly, the midpoint potential of the E3 FAD shifts dramatically to -273 mV (E1o' approximately -345 mV and E2o' approximately -200 mV) at pH 8.4, with very little semiquinone stabilization (< 5%). The potential of the E3 [2Fe-2S] center at pH 8.4 was also found to undergo a negative shift to -279 mV, and that of the E1 iron sulfur center remained essentially the same at -206 mV. These data indicated that the redox properties of this system may be regulated by pH and the electron transfer between the E3 redox centers may be prototropically controlled. These results also demonstrated that E3 is unique among this class of enzymes.
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PMID:Studies of the redox properties of CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1) and CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3): two important enzymes involved in the biosynthesis of ascarylose. 867 89

The lipopolysaccharide of Yersinia pseudotuberculosis V includes a 3,6-dideoxyhexose, ascarylose, as the nonreducing end of the O-antigen tetrasaccharide. The C-3 deoxygenation of CDP-6-deoxy-L-threo-D-glycero-4-hexulose is a critical reaction in the biosynthesis of ascarylose. The first half of the reaction is a dehydration catalyzed by CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), which is PMP-dependent and contains a redox-active [2Fe-2S] center. The second half is a reduction that requires an additional enzyme, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3, formerly known as CDP-6-deoxy-delta 3,4-glucoseen reductase), which has a FAD and a [2Fe-2S] center in the active site. Using NADH as the reductant in the coupled E1-E3 reaction, we have monitored the kinetics of a radical intermediate using both stopped-flow spectrophotometry and rapid freeze-quench EPR under aerobic and hypoxic conditions. In the EPR studies, a sharp signal at g = 2.003 was found to appear at a rate which is kinetically competent, reaching its maximum intensity at approximately 150 ms. Stopped-flow UV-vis analysis of the reaction elucidated a minimum of six optically distinguishable states in the mechanism of electron transfer from NADH to substrate. Interestingly, one of the detected intermediates has a time course nearly identical to that of the radical detected by rapid freeze-quench EPR. The difference UV-vis spectrum of this intermediate displays a maximum at 456 nm with a shoulder at 425 nm. Overall, these results are consistent with an electron transfer pathway that includes a radical intermediate with the unpaired spin localized on the substrate-cofactor complex. Evidence in support of this mechanism is presented in this report. These studies add the PMP-glucoseen radical to the growing list of mechanistically important bioorganic radical intermediates that have recently been discovered.
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PMID:Kinetic characterization of an organic radical in the ascarylose biosynthetic pathway. 896 49