Further characterization of two different,reversible aldehyde oxidoreductases from Clostridium formicoaceticum,one containing tungsten and the other molybdenum

The tungsten- and the molybdenum-containing aldehyde oxidoreductases from Clostridium formicoaceticum show, for aldehydes, K _m values<30 M and K _i values of millimolar concentrations. The tungsten-containing aldehyde oxidoreductase is inactivated to 50% by 3 mM KCN within 1 min, by 1 mM ferricyanide within 5 min, and by 0.05 mM chloralhydrate within 30 s. The molybdenum-containing AOR shows 50% inactivation within 1 min only with 70 mM KCN. The tungsten-containing enzyme is very sensitive to oxygen, especially in the reduced state, whereas the molybdenum-containing enzyme exhibits only moderate oxygen sensitivity without being markedly influenced by the redox state of the enzyme. The tungsten in the aldehyde oxidoreductase is bound to a pterin cofactor (Wco) of the mononucleotide form that is known for molybdopterin cofactor (Moco). The nature of the molybdenum cofactor in the molybdenum-containing aldehyde oxidoreductase is still unclear. The UV/VIS spectrum of the tungsten-containing aldehyde oxidoreductase shows a broad absorption in the range of 400 nm with a millimolar absorption coefficient of 18.1 (reduced form) and 24.8 (dehydrogenated form) at 396 nm. The epr spectrum exhibits two different W(V) signals with the following g values for signal A: 2.035, 1.959, 1.899 and signal B: 2.028, 2.017, 2.002. Dithionite-reduced enzyme shows signals of 4Fe–4S or 2Fe–2S clusters. Initial rate studies with different substrates for the carboxylate reduction led to a Bi Uni Uni Bi mechanism.Abbreviations AOR aldehyde oxidoreductase - NH ₂ CO-MV 1,1-carbamoylmethylviologen - MV methylviologen - TMV 1,1,2,2-tetramethylviologen     

Metabolic engineering of Clostridium autoethanogenum for selective alcohol production

Gas fermentation using acetogenic bacteria such as Clostridium autoethanogenum offers an attractive route for production of fuel ethanol from industrial waste gases. Acetate reduction to acetaldehyde and further to ethanol via an aldehyde: ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase has been postulated alongside the classic pathway of ethanol formation via a bi-functional aldehyde/alcohol dehydrogenase (AdhE). Here we demonstrate that AOR is critical to ethanol formation in acetogens and inactivation of AdhE led to consistently enhanced autotrophic ethanol production (up to 180%). Using ClosTron and allelic exchange mutagenesis, which was demonstrated for the first time in an acetogen, we generated single mutants as well as double mutants for both aor and adhE isoforms to confirm the role of each gene. The aor1+2 double knockout strain lost the ability to convert exogenous acetate, propionate and butyrate into the corresponding alcohols, further highlighting the role of these enzymes in catalyzing the thermodynamically unfavourable reduction of carboxylic acids into alcohols.     

Pterin cofactor,substrate specificity,and observations on the kinetics of the reversible tungsten-containing aldehyde oxidoreductase fromClostridium thermoaceticum

The αβ subunits of the tungsten-containing reversible aldehyde oxidoreductase of Clostridium thermoaceticum were shown to contain a pterin cofactor in the form of a mononucleotide. The substrate specificity of the enzyme for aliphatic and aromatic aldehydes and for carboxylates was broad. The K _m values for ethanal, propanal and butanal were 0.010–0.006 mM, but the value for methanal was 1.6 mM. Benzaldehyde derivatives with a hydroxy group in the 4-position showed millimolar K _m values that were 2–3 orders of magnitude higher than those of other aromatic and aliphatic aldehydes. The ratio of k _cat /K _m for aldehydes and the corresponding acids is 10⁴–10⁵. For carboxylate reduction, 4-hydroxy benzoate again showed the highest K _m value of all substrates tested. When the 4-hydroxy groups of the aldehyde and the acid were methylated, the K _m values were decreased drastically. From the temperature dependence of carboxylate reduction at the expense of viologens, activation energies that depended on the substrate and on the applied viologen were calculated. The pH optima of the carboxylate reductions depended on the pK values of the acids and shifted to lower pH values with lower pK values of the acids. The ternary complex α₃β₃γ of the aldehyde oxidoreductase was able to dehydrogenate aldehydes to acylates with NADP⁺. Surprisingly the reverse reaction was observed too, although at very low rates. When exposed to air, the aldehyde oxidoreductase showed markedly enhanced lability in its reduced state compared to its oxidized state. With resting cells of C. thermoaceticum, many carboxylates were reduced at the expense of carbon monoxide to the corresponding alcohols. Received: 18 January 1995 / Accepted: 5 April 1995     

Purification and Molecular Characterization of the Tungsten-Containing Formaldehyde Ferredoxin Oxidoreductase from the Hyperthermophilic Archaeon Pyrococcus furiosus: the Third of a Putative Five-Member Tungstoenzyme Family

Pyrococcus furiosus is a hyperthermophilic archaeon which grows optimally near 100°C by fermenting peptides and sugars to produce organic acids, CO₂, and H₂. Its growth requires tungsten, and two different tungsten-containing enzymes, aldehyde ferredoxin oxidoreductase (AOR) and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR), have been previously purified from P. furiosus. These two enzymes are thought to function in the metabolism of peptides and carbohydrates, respectively. A third type of tungsten-containing enzyme, formaldehyde ferredoxin oxidoreductase (FOR), has now been characterized. FOR is a homotetramer with a mass of 280 kDa and contains approximately 1 W atom, 4 Fe atoms, and 1 Ca atom per subunit, together with a pterin cofactor. The low recovery of FOR activity during purification was attributed to loss of sulfide, since the purified enzyme was activated up to fivefold by treatment with sulfide (HS⁻) under reducing conditions. FOR uses P. furiosus ferredoxin as an electron acceptor (K_m = 100 μM) and oxidizes a range of aldehydes. Formaldehyde (K_m = 15 mM for the sulfide-activated enzyme) was used in routine assays, but the physiological substrate is thought to be an aliphatic C₅ semi- or dialdehyde, e.g., glutaric dialdehyde (K_m = 1 mM). Based on its amino-terminal sequence, the gene encoding FOR (for) was identified in the genomic database, together with those encoding AOR and GAPOR. The amino acid sequence of FOR corresponded to a mass of 68.7 kDa and is highly similar to those of the subunits of AOR (61% similarity and 40% identity) and GAPOR (50% similarity and 23% identity). The three genes are not linked on the P. furiosus chromosome. Two additional (and nonlinked) genes (termed wor4 and wor5) that encode putative tungstoenzymes with 57% (WOR4) and 56% (WOR5) sequence similarity to FOR were also identified. Based on sequence motif similarities with FOR, both WOR4 and WOR5 are also proposed to contain a tungstobispterin site and one [4Fe-4S] cluster per subunit.     

萝卜TALE转录因子家族的鉴定与分析

TALE (three-amino acid loop extension)转录因子在植物生长发育及细胞分化过程中起重要作用.在多种植物中均已鉴定出TALE转录因子的家族成员,但是萝卜TALE转录因子家族的研究鲜有报道.文中通过生物信息学手段在象牙白萝卜全基因组中鉴定出了分布于9条染色体上的33个TALE家族基因.研究...     

The tungsten metallome of Pyrococcus furiosus

The tungsten metallome of the hyperthermophilic archaeon Pyrococcus furiosus has been investigated using electroanalytical metal analysis and native-native 2D-PAGE with the radioactive tungsten isotope (187)W (t(1/2) = 23.9 h). P. furiosus cells have an intracellular tungsten concentration of 29 μM, of which ca. 30% appears to be free tungsten, probably in the form of tungstate or polytungstates. The remaining 70% is bound by five different tungsten enzymes: formaldehyde ferredoxin oxidoreductase, aldehyde ferredoxin oxidoreductase, glyceraldehyde-3-phosphate ferredoxin oxidoreductase and the tungsten-containing oxidoreductases WOR4 and WOR5. The membrane proteome of P. furiosus is devoid of tungsten. The differential expression, as measured by the tungsten level, of the five soluble tungsten enzymes when the cells are subjected to a cold-shock shows a strong correlation with previously published DNA microarray analyses.