Hydroxylamine reductase activity of the hybrid cluster protein from Escherichia coli

The hybrid cluster protein (HCP; formerly termed the prismane protein) has been extensively studied due to its unique spectroscopic properties. Although the structural and spectroscopic characteristics are well defined, its enzymatic function, up to this point, has remained unidentified. While it was proposed that HCP acts in some step of nitrogen metabolism, a specific role for this enzyme remained unknown. Recent studies of HCP purified from Escherichia coli have identified a novel hydroxylamine reductase activity. These data reveal the ability of HCP to reduce hydroxylamine in vitro to form NH(3) and H(2)O. Further biochemical analyses were completed in order to determine the effects of various electron donors, different pH levels, and the presence of CN(-) on in vitro hydroxylamine reduction.     

YqhD: a broad-substrate range aldehyde reductase with various applications in production of biorenewable fuels and chemicals

The Escherichia coli NADPH-dependent aldehyde reductase YqhD has contributed to a variety of metabolic engineering projects for production of biorenewable fuels and chemicals. As a scavenger of toxic aldehydes produced by lipid peroxidation, YqhD has reductase activity for a broad range of short-chain aldehydes, including butyraldehyde, glyceraldehyde, malondialdehyde, isobutyraldehyde, methylglyoxal, propanealdehyde, acrolein, furfural, glyoxal, 3-hydroxypropionaldehyde, glycolaldehyde, acetaldehyde, and acetol. This reductase activity has proven useful for the production of biorenewable fuels and chemicals, such as isobutanol and 1,3- and 1,2-propanediol; additional capability exists for production of 1-butanol, 1-propanol, and allyl alcohol. A drawback of this reductase activity is the diversion of valuable NADPH away from biosynthesis. This YqhD-mediated NADPH depletion provides sufficient burden to contribute to growth inhibition by furfural and 5-hydroxymethyl furfural, inhibitory contaminants of biomass hydrolysate. The structure of YqhD has been characterized, with identification of a Zn atom in the active site. Directed engineering efforts have improved utilization of 3-hydroxypropionaldehyde and NADPH. Most recently, two independent projects have demonstrated regulation of yqhD by YqhC, where YqhC appears to function as an aldehyde sensor.     

Substrate-induced hysteresis in the activity of Escherichia coli dihydrofolate reductase

Full time course studies of the kinetic activity of Escherichia coli dihydrofolate reductase show that there is an increase in activity with time. The half-time for this hysteretic behavior is about 9 s. Preincubation of the enzyme with either of the substrates abolishes the lag and results in initial velocities which are 2-2.3-fold faster than those observed for the non-preincubated enzyme. The kinetic properties of the activated and nonactivated forms of the enzyme appear to be similar as measured by the full time course of the reaction. The results are consistent with observations for NADPH binding studies that the enzyme exists in two interconvertible forms, one of which is incapable of binding NADPH (Cayley, P. J., Dunn, S. M. J., and King, R. W. (1981) Biochemistry 20, 874-879).     

Folding of dihydrofolate reductase from Escherichia coli

The urea-induced equilibrium unfolding transition of dihydrofolate reductase from Escherichia coli was monitored by UV difference, circular dichroism (CD), and fluorescence spectroscopy. Each of these data sets were well described by a two-state unfolding model involving only native and unfolded forms. The free energy of folding in the absence of urea at pH 7.8, 15 degrees C is 6.13 +/- 0.36 kcal mol-1 by difference UV, 5.32 +/- 0.67 kcal mol-1 by CD, and 5.42 +/- 1.04 kcal mol-1 by fluorescence spectroscopy. The midpoints for the difference UV, CD, and fluorescence transitions are 3.12, 3.08, and 3.18 M urea, respectively. The near-coincidence of the unfolding transitions monitored by these three techniques also supports the assignment of a two-state model for the equilibrium results. Kinetic studies of the unfolding and refolding reactions show that the process is complex and therefore that additional species must be present. Unfolding jumps in the absence of potassium chloride revealed two slow phases which account for all of the amplitude predicted by equilibrium experiments. Unfolding in the presence of 400 mM KCl results in the selective loss of the slower phase, implying that there are two native forms present in equilibrium prior to unfolding. Five reactions were observed in refolding: two slow phases designated tau 1 and tau 2 that correspond to the slow phases in unfolding and three faster reactions designated tau 3, tau 4, and tau 5 that were followed by stopped-flow techniques. The kinetics of the recovery of the native form was monitored by following the binding of methotrexate, a tight-binding inhibitor of dihydrofolate reductase, at 380 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Channel-closing activity of porins from Escherichia coli in bilayer lipid membranes

The opening and closing of the ompF porin from Escherichia coli JF 701 was investigated by reconstituting the purified protein into planar bilayer membranes. The electrical conductance changes across the membranes at constant potential were used to analyze the size and aggregate nature of the porin channel complexes and the relative number of opening and closing events. We found that, when measured at pH 5.5, the channel conductance diminished and the number of closing events increased when the voltage was greater than 100 mV. The results suggest that the number of smaller sized conductance channels increases above this potential. There was also an increase in the smaller subunits and in the closing events when the pH was lowered to 3.5, and these changes were further enhanced by increasing the voltage. We propose that both lowering the pH and elevating the potential across the membrane stabilize the porin in a conformation in which the subunits are less tightly associated and the subunits open in a non-cooperative manner. These same conditions also appear to stabilize the closed state of the pore.     

Reduction of methylglyoxal in Escherichia coli K12 by an aldehyde reductase and alcohol dehydrogenase

Two enzymes, one NADPH-dependent and another NADH-dependent which catalyze the reduction of methylglyoxal to acetol have been isolated and substantially purified from crude extracts of Escherichia coli K₁₂ cells. Substrate specificity and formation of acetol as the reaction product by both the enzymes, reversibility of NADH-dependent enzyme with alcohols as substrates and inhibitor study with NADPH-dependent enzyme indicate that NADPH-dependent and NADH-dependent enzymes are identical with an aldehyde reductase (EC 1.1.1.2) and alcohol dehydrogenase (EC 1.1.1.1) respectively. The K_m for methylglyoxal have been determined to be 0.77 mM for NADPH-dependent and 3.8 mM for NADH-dependent enzyme. Stoichiometrically equimolar amount of acetol is formed from methylglyoxal by both NADPH- and NADH-dependent enzymes. In phosphate buffer, both the enzymes are active in the pH range of 5.8–6.6 with no sharp pH optimum. Molecular weight of both the enzymes were found to be 100,000 ± 3,000 by gel filtration on a Sephacryl S-200 column. Both NADPH- and NADH-dependent enzymes are sensitive to sulfhydryl group reagents.     

Characterization of an NADH-linked cupric reductase activity from the Escherichia coli respiratory chain.

Previous results from our laboratory have shown that NADH-supported electron flow through the Escherichia coli respiratory chain promotes the reduction of cupric ions to Cu(I), which mediates damage of the respiratory system by hydroperoxides. The aim of this work was to characterize the NADH-linked cupric reductase activity from the E. coli respiratory chain. We have used E. coli strains that either overexpress or are deficient in the NADH dehydrogenase-2 (NDH-2) to demonstrate that this membrane-bound protein catalyzes the electron transfer from NADH to Cu(II), but not to Fe(III). We also show that purified NDH-2 exhibits NADH-supported Cu(II) reductase activity in the presence of either FAD or quinone, but is unable to reduce Fe(III). The K(m) values for free Cu(II) were 32 +/- 5 pM in the presence of saturating duroquinone and 22 +/- 2 pM in the presence of saturating FAD. The K(m) values for NADH were 6.9 +/- 1.5 microM and 6.1 +/- 0.7 microM in the presence of duroquinone and FAD, respectively. The quinone-dependent Cu(II) reduction occurred through both O(*-)(2)-mediated and O(*-)(2)-independent pathways, as evidenced by the partial inhibitory effect (30-50%) of superoxide dismutase, by the reaction stoichiometry, and by the enzyme turnover numbers for NADH and Cu(II). The cupric reductase activity of NDH-2 was dependent on thiol groups which were accessible to p-chloromercuribenzoate at low, but not at high, ionic strength of the medium, a fact apparently connected to a conformational change of the protein. To our knowledge, this is the first protein with cupric reductase activity to be isolated and characterized in its biochemical properties.     

Succinate dehydrogenase and fumarate reductase from Escherichia coli

Succinate-ubiquinone oxidoreductase (SQR) as part of the trichloroacetic acid cycle and menaquinol-fumarate oxidoreductase (QFR) used for anaerobic respiration by Escherichia coli are structurally and functionally related membrane-bound enzyme complexes. Each enzyme complex is composed of four distinct subunits. The recent solution of the X-ray structure of QFR has provided new insights into the function of these enzymes. Both enzyme complexes contain a catalytic domain composed of a subunit with a covalently bound flavin cofactor, the dicarboxylate binding site, and an iron–sulfur subunit which contains three distinct iron–sulfur clusters. The catalytic domain is bound to the cytoplasmic membrane by two hydrophobic membrane anchor subunits that also form the site(s) for interaction with quinones. The membrane domain of E. coli SQR is also the site where the heme b₅₅₆ is located. The structure and function of SQR and QFR are briefly summarized in this communication and the similarities and differences in the membrane domain of the two enzymes are discussed.     

A novel aldose/aldehyde reductase protects transgenic plants against lipid peroxidation under chemical and drought stresses

Rapid accumulation of toxic products from reactions of reactive oxygen species (ROS) with lipids and proteins significantly contributes to the damage of crop plants under biotic and abiotic stresses. Here we have identified a stress-activated alfalfa gene encoding a novel plant NADPH-dependent aldose/aldehyde reductase that also exhibited characteristics of the homologous human enzyme. The recombinant alfalfa enzyme is active on 4-hydroxynon-2-enal, a known cytotoxic lipid peroxide degradation product. Ectopic synthesis of this enzyme in transgenic tobacco plants provided considerable tolerance against oxidative damage caused by paraquat and heavy metal treatment. These transformants could also resist a long period of water deficiency and exhibited improved recovery after rehydration. We found a reduced production of lipid peroxidation-derived reactive aldehydes in these transformed plants under different stresses. These studies reveal a new and efficient detoxification pathway in plants.     

Dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli: dihydrofolate monooxygenase activity

Dihydrofolate reductase from strain MB 1428 of Escherichia coli was shown to catalyze the oxidative cleavage of dihydrofolate at the C(9)N(10) bond. One of the products of the reaction was identified as 7,8-dihydropterin-6-carboxaldehyde through its proton magnetic resonance spectrum. The maximal enzymatic rate was 0.05 moles dihydrofolate cleaved per minute per mole enzyme at 25° and pH 7.2, and the K_M for dihydrofolate was 17.5 ± 2.5 μM. The enzymatic reaction was fully inhibitable with methotrexate. The mechanism of enzyme action was proposed to be an apparent “acidification” of dihydrofolate upon binding to the enzyme. Folate underwent an analogous oxidative cleavage by enzyme with a turnover number of 0.0014, which produced pterin-6-carboxaldehyde. Methotrexate was also slowly degraded by the enzyme.     

ATPase activity of the MsbA lipid flippase of Escherichia coli

Escherichia coli MsbA, the proposed inner membrane lipid flippase, is an essential ATP-binding cassette transporter protein with homology to mammalian multidrug resistance proteins. Depletion or loss of function of MsbA results in the accumulation of lipopolysaccharide and phospholipids in the inner membrane of E. coli. MsbA modified with an N-terminal hexahistidine tag was overexpressed, solubilized with a nonionic detergent, and purified by nickel affinity chromatography to approximately 95% purity. The ATPase activity of the purified protein was stimulated by phospholipids. When reconstituted into liposomes prepared from E. coli phospholipids, MsbA displayed an apparent K(m) of 878 microm and a V(max) of 37 nmol/min/mg for ATP hydrolysis in the presence of 10 mm Mg(2+). Preincubation of MsbA-containing liposomes with 3-deoxy-d-mannooctulosonic acid (Kdo)(2)-lipid A increased the ATPase activity 4-5-fold, with half-maximal stimulation seen at 21 microm Kdo(2)-lipid A. Addition of Kdo(2)-lipid A increased the V(max) to 154 nmol/min/mg and decreased the K(m) to 379 microm. Stimulation was only seen with hexaacylated lipid A species and not with precursors, such as diacylated lipid X or tetraacylated lipid IV(A). MsbA containing the A270T substitution, which renders cells temperature-sensitive for growth and lipid export, displayed ATPase activity similar to that of the wild type protein at 30 degrees C but was significantly reduced at 42 degrees C. These results provide the first in vitro evidence that MsbA is a lipid-activated ATPase and that hexaacylated lipid A is an especially potent activator.     

Site-specific mutagenesis of dihydrofolate reductase from Escherichia coli

Two site-specific mutations of dihydrofolate reductase from Escherichia coli based on the x-ray crystallographic structure were constructed. The first mutation (His-45----Gln) is aimed at assessing the interaction between the imidazole moiety and the pyrophosphate backbone of NADPH. The second (Thr-113----Val) is part of a hydrogen bonding network that contacts the dihydrofolate substrate and may be involved in proton delivery to the N5-C6 imine undergoing reduction. The first mutation was shown to alter both the association and dissociation rate constants for the cofactor so that the dissociation constant was increased 6-40-fold. A corresponding but smaller (fourfold) effect was noted in V/K but not in V compared to the wild-type enzyme. The second was demonstrated to increase the dissociation rate constant for methotrexate 20-30-fold, and presumably dihydrofolate also, with a corresponding 20-30-fold increase in the dissociation constant. In this case an identical effect was noted on V/K but not in V relative to the native enzyme. Thus, in both mutant enzymes the decrease in binding has not been translated into a loss of catalytic efficiency.     

Novel inhibitors of beta-ketoacyl-ACP reductase from Escherichia coli

Bacterial beta-ketoacyl-[acyl carrier protein] (beta-ketoacyl-ACP) reductase (FabG) is a highly conserved and ubiquitously expressed enzyme of the fatty-acid biosynthetic pathway of prokaryotic organisms that catalyzes NADPH-dependent reduction of beta-ketoacyl-ACP intermediates. Therefore, FabG represents an appealing target for the development of new antimicrobial agents. A number of trans-cinnamic acid derivatives were designed and screened for inhibitory activities against FabG from Escherichia coli. These inhibited FabG enzymatic activity with IC(50) values in the microM range, and were used as templates for the subsequent diversification of the chemotype. Introduction of an electron-withdrawing 4-cyano group to the phenol substituent showed improved inhibition over the non-substituted compound. The benzo-[1,3]-dioxol moiety also appeared to be essential for inhibitory activity of trans-cinnamic acid derivatives against FabG from E. coli. To explain the possible binding position, the best inhibitor from the present study was docked in the active site of FabG. The results for the best scoring conformers chosen by the docking programme revealed that cinnamic acid derivatives can be accommodated in the substrate-binding region of the active site, above the nicotinamide moiety of the NADPH cofactor. Additionally, a phage-displayed library of random linear 15-mer peptides was screened against FabG, to identify ligands with the common PPLTXY motif.     

Succinate dehydrogenase and fumarate reductase from Escherichia coli.

Succinate-ubiquinone oxidoreductase (SQR) as part of the trichloroacetic acid cycle and menaquinol-fumarate oxidoreductase (QFR) used for anaerobic respiration by Escherichia coli are structurally and functionally related membrane-bound enzyme complexes. Each enzyme complex is composed of four distinct subunits. The recent solution of the X-ray structure of QFR has provided new insights into the function of these enzymes. Both enzyme complexes contain a catalytic domain composed of a subunit with a covalently bound flavin cofactor, the dicarboxylate binding site, and an iron-sulfur subunit which contains three distinct iron-sulfur clusters. The catalytic domain is bound to the cytoplasmic membrane by two hydrophobic membrane anchor subunits that also form the site(s) for interaction with quinones. The membrane domain of E. coli SQR is also the site where the heme b556 is located. The structure and function of SQR and QFR are briefly summarized in this communication and the similarities and differences in the membrane domain of the two enzymes are discussed.     

Purification and characterization of an azoreductase from Escherichia coli CD-2 possessing quinone reductase activity

An oxygen-insensitive intracellular enzyme that is responsible for the decolorization of azo dyes was purified from Escherichia coli CD-2. The molecular weight of the purified enzyme was estimated as 27,000 ± 500 Da. Protein identification indicated that the enzyme had high sequence homology with E. coli K12 quinone reductase, and the enzyme was proved to have both azoreductase and quinone reductase activity. With methyl red as substrate, the optimal pH value and temperature were 6.5 and 37 °C, respectively. The enzyme was stable under different physiochemical conditions. The azoreductase activity was restrained by SDS and was almost completely inhibited by Co²⁺ and Hg²⁺. K_m and V_max values were 0.18 mM and 8.12 U mg^?1 of protein for NADH and 0.05 mM and 6.46 U mg^?1 of protein for methyl red, respectively. The purified enzyme could efficiently decolorize methyl red with both NADH and NADPH as electron donors.