Publications

• Dehydration is catalyzed by glutamate-136 and aspartic acid-135 active site residues in Escherichia coli dTDP-glucose 4,6-dehydratase.

  Gross J.W., Hegeman A.D., Gerratana B., Frey P.A.

  The dTDP-glucose 4,6-dehydratase catalyzed conversion of dTDP-glucose to dTDP-4-keto-6-deoxyglucose occurs in three sequential chemical steps: dehydrogenation, dehydration, and rereduction. The enzyme contains the tightly bound coenzyme NAD(+), which mediates the dehydrogenation and rereduction st ... >> More

  The dTDP-glucose 4,6-dehydratase catalyzed conversion of dTDP-glucose to dTDP-4-keto-6-deoxyglucose occurs in three sequential chemical steps: dehydrogenation, dehydration, and rereduction. The enzyme contains the tightly bound coenzyme NAD(+), which mediates the dehydrogenation and rereduction steps of the reaction mechanism. In this study, we have determined that Asp135 and Glu136 are the acid and base catalysts, respectively, of the dehydration step. Identification of the acid catalyst was performed using an alternative substrate, dTDP-6-fluoro-6-deoxyglucose (dTDP-6FGlc), which undergoes fluoride ion elimination instead of dehydration, and thus does not require protonation of the leaving group. The steady-state rate of conversion of dTDP-6FGlc to dTDP-4-keto-6-deoxyglucose by each Asp135 variant was identical to that of wt, in contrast to turnover using dTDP-glucose where differences in rates of up to 2 orders of magnitude were observed. These results demonstrate Asp135's role in protonating the glucosyl-C6(OH) during dehydration. The base catalyst was identified using a previously uncharacterized, enzyme-catalyzed glucosyl-C5 hydrogen-solvent exchange reaction of product, dTDP-4-keto-6-deoxyglucose. Base catalysis of this exchange reaction is analogous to that occurring at C5 during the dehydration step of net catalysis. Thus, the decrease in the rate of catalysis ( approximately 2 orders of magnitude) of the exchange reaction observed with Glu136 variants demonstrates this residue's importance in base catalysis of dehydration. << Less

  Biochemistry 40:12497-12504(2001) [PubMed] [EuropePMC]

• Characterization of a novel thermostable Mn(II)-dependent 2,3-dihydroxybiphenyl 1,2-dioxygenase from a polychlorinated biphenyl- and naphthalene-degrading Bacillus sp. JF8.

  Hatta T., Mukerjee-Dhar G., Damborsky J., Kiyohara H., Kimbara K.

  A novel thermostable Mn(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC_JF8) catalyzing the meta-cleavage of the hydroxylated biphenyl ring was purified from the thermophilic biphenyl and naphthalene degrader, Bacillus sp. JF8, and the gene was cloned. The native and recombinant BphC enz ... >> More

  A novel thermostable Mn(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC_JF8) catalyzing the meta-cleavage of the hydroxylated biphenyl ring was purified from the thermophilic biphenyl and naphthalene degrader, Bacillus sp. JF8, and the gene was cloned. The native and recombinant BphC enzyme was purified to homogeneity. The enzyme has a molecular mass of 125 +/-10 kDa and was composed of four identical subunits (35 kDa). BphC_JF8 has a temperature optimum of 85 degrees C and a pH optimum of 7.5. It exhibited a half-life of 30 min at 80 degrees C and 81 min at 75 degrees C, making it the most thermostable extradiol dioxygenase studied. Inductively coupled plasma mass spectrometry analysis confirmed the presence of 4.0-4.8 manganese atoms per enzyme molecule. The EPR spectrum of BphC_JF8 exhibited g = 2.02 and g = 4.06 signals having the 6-fold hyperfine splitting characteristic of Mn(II). The enzyme can oxidize a wide range of substrates, and the substrate preference was in the order 2,3-dihydroxybiphenyl > 3-methylcatechol > catechol > 4-methylcatechol > 4-chlorocatechol. The enzyme is resistant to denaturation by various chelators and inhibitors (EDTA, 1,10-phenanthroline, H2O2, 3-chlorocatechol) and did not exhibit substrate inhibition even at 3 mm 2,3-dihydroxybiphenyl. A decrease in Km accompanied an increase in temperature, and the Km value of 0.095 microm for 2,3-dihydroxybiphenyl (at 60 degrees C) is among the lowest reported. The kinetic properties and thermal stability of the native and recombinant enzyme were identical. The primary structure of BphC_JF8 exhibits less than 25% sequence identity to other 2,3-dihydroxybiphenyl 1,2-dioxygenases. The metal ligands and active site residues of extradiol dioxygenases are conserved, although several amino acid residues found exclusively in enzymes that preferentially cleave bicyclic substrates are missing in BphC_JF8. A three-dimensional homology model of BphC_JF8 provided a basis for understanding the substrate specificity, quaternary structure, and stability of the enzyme. << Less

  J. Biol. Chem. 278:21483-21492(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Probing catalysis by Escherichia coli dTDP-glucose-4,6-dehydratase: identification and preliminary characterization of functional amino acid residues at the active site.

  Hegeman A.D., Gross J.W., Frey P.A.

  A model of the Escherichia coli dTDP-glucose-4,6-dehydratase (4,6-dehydratase) active site has been generated by combining amino acid sequence alignment information with the 3-dimensional structure of UDP-galactose-4-epimerase. The active site configuration is consistent with the partially refined ... >> More

  A model of the Escherichia coli dTDP-glucose-4,6-dehydratase (4,6-dehydratase) active site has been generated by combining amino acid sequence alignment information with the 3-dimensional structure of UDP-galactose-4-epimerase. The active site configuration is consistent with the partially refined 3-dimensional structure of 4,6-dehydratase, which lacks substrate-nucleotide but contains NAD(+) (PDB file ). From the model, two groups of active site residues were identified. The first group consists of Asp135(DEH), Glu136(DEH), Glu198(DEH), Lys199(DEH), and Tyr301(DEH). These residues are near the substrate-pyranose binding pocket in the model, they are completely conserved in 4,6-dehydratase, and they differ from the corresponding equally well-conserved residues in 4-epimerase. The second group of residues is Cys187(DEH), Asn190(DEH), and His232(DEH), which form a motif on the re face of the cofactor nicotinamide binding pocket that resembles the catalytic triad of cysteine-proteases. The importance of both groups of residues was tested by mutagenesis and steady-state kinetic analysis. In all but one case, a decrease in catalytic efficiency of approximately 2 orders of magnitude below wild-type activity was observed. Mutagenesis of each of these residues, with the exception of Cys187(DEH), which showed near-wild-type activity, clearly has important negative consequences for catalysis. The allocation of specific functions to these residues and the absolute magnitude of these effects are obscured by the complex chemistry in this multistep mechanism. Tools will be needed to characterize each chemical step individually in order to assign loss of catalytic efficiency to specific residue functions. To this end, the effects of each of these variants on the initial dehydrogenation step were evaluated using a the substrate analogue dTDP-xylose. Additional steady-state techniques were employed in an attempt to further limit the assignment of rate limitation. The results are discussed within the context of the 4,6-dehydratase active site model and chemical mechanism. << Less

  Biochemistry 40:6598-6610(2001) [PubMed] [EuropePMC]

• Toward a structural understanding of the dehydratase mechanism.

  Allard S.T., Beis K., Giraud M.-F., Hegeman A.D., Gross J.W., Wilmouth R.C., Whitfield C., Graninger M., Messner P., Allen A.G., Maskell D.J., Naismith J.H.

  dTDP-D-glucose 4,6-dehydratase (RmlB) was first identified in the L-rhamnose biosynthetic pathway, where it catalyzes the conversion of dTDP-D-glucose into dTDP-4-keto-6-deoxy-D-glucose. The structures of RmlB from Salmonella enterica serovar Typhimurium in complex with substrate deoxythymidine 5' ... >> More

  dTDP-D-glucose 4,6-dehydratase (RmlB) was first identified in the L-rhamnose biosynthetic pathway, where it catalyzes the conversion of dTDP-D-glucose into dTDP-4-keto-6-deoxy-D-glucose. The structures of RmlB from Salmonella enterica serovar Typhimurium in complex with substrate deoxythymidine 5'-diphospho-D-glucose (dTDP-D-glucose) and deoxythymidine 5'-diphosphate (dTDP), and RmlB from Streptococcus suis serotype 2 in complex with dTDP-D-glucose, dTDP, and deoxythymidine 5'-diphospho-D-pyrano-xylose (dTDP-xylose) have all been solved at resolutions between 1.8 A and 2.4 A. The structures show that the active sites are highly conserved. Importantly, the structures show that the active site tyrosine functions directly as the active site base, and an aspartic and glutamic acid pairing accomplishes the dehydration step of the enzyme mechanism. We conclude that the substrate is required to move within the active site to complete the catalytic cycle and that this movement is driven by the elimination of water. The results provide insight into members of the SDR superfamily. << Less

  Structure 10:81-92(2002) [PubMed] [EuropePMC]

• The mechanism of 6-deoxyhexose synthesis. I. Intramolecular hydrogen transfer catalyzed by deoxythymidine diphosphate D-glucose oxidoreductase.

  Melo A., Elliott W.H., Glaser L.

  J Biol Chem 243:1467-1474(1968) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.