Involvement of lactaldehyde dehydrogenase in several metabolic pathways of Escherichia coli K12

Lactaldehyde dehydrogenase (E.C. 1.2.1.22) of Escherichia coli has been purified to homogeneity. It has four apparently equal subunits (molecular weight 55,000 each) and four NAD binding sites per molecule of native enzyme. The enzyme is inducible, only under aerobic conditions, by at least three different types of molecules, the sugars fucose and rhamnose, the diol ethylene glycol and the amino acid glutamate. The enzyme catalyzes the irreversible oxidation of several aldehydes with a Km in the micromolar range for alpha-hydroxyaldehydes (lactaldehyde, glyceraldehyde, or glycolaldehyde) and a higher Km, in the millimolar range, for the alpha-ketoaldehyde methylglyoxal. It displays substrate inhibition with all these substrates. NAD is the preferential cofactor. The functional and structural features of the enzyme indicate that it is not an isozyme of other E. coli aldehyde dehydrogenases such as glyceraldehyde phosphate dehydrogenase, glycolaldehyde dehydrogenase, or acetaldehyde dehydrogenase. The enzyme, previously described as specific for lactaldehyde, is thus identified as a dehydrogenase with a fairly general role in aldehyde oxidation, and it is probably involved in several metabolic pathways.     

Chlorate Reducing Activity of Spinach Nitrate Reductase

The activities of 2-oxoaldehyde-metabolizing enzymes (glyoxalase I, glyoxalase II, methyl- glyoxal reductase, methylglyoxal dehydrogenase and lactaldehyde dehydrogenase) were found to be widely distributed among microorganisms. One of the enzymes, methylglyoxal reductase, which catalyzes the reductive conversion of methylglyoxal into lactaldehyde, was purified from Escherichia coli cells. The enzyme was judged to be homogeneous on polyacrylamide gel electrophoresis and was a monomer with a molecular weight of 43000. The enzyme was most active at pH 6.5 and 45°C. The enzyme utilized both NADPH and NADH for the reduction of 2- oxoaldehydes (glyoxal, methylglyoxal, phenylglyoxal and 4,5-dioxovalerate) and some aldehydes (glycolaldehyde, D,l-glyceraldehyde, propionaldehyde and acetaldehyde). The Km values of the enzyme for methylglyoxal, NADPH and NADH were 4.0 mm, 1.7 fiM and 2.8 /¿m, respectively. The product of methylglyoxal reduction was identified as lactaldehyde. The enzyme from E. coli cells was different from the yeast and goat liver enzymes in both molecular structure and substrate specificity.     

Experimental evolution of a metabolic pathway for ethylene glycol utilization by Escherichia coli.

Spontaneous mutants of Escherichia coli able to grow on ethylene glycol as a sole source of carbon and energy were obtained from mutants that could grow on propylene glycol. Attempts to obtain ethylene glycol-utilizing mutants from wild-type E. coli were unsuccessful. The two major characteristics of the ethylene glycol-utilizing mutants were (i) increased activities of propanediol oxidoreductase, an enzyme present in the parental strain (a propylene glycol-positive strain), which also converted ethylene glycol into glycolaldehyde; and (ii) constitutive synthesis of high activities of glycolaldehyde dehydrogenase, which converted glycolaldehyde to glycolate. Glycolate was metabolized via the glycolate pathway, which was present in the wild-type cells; this was indicated by the induction in ethylene glycol-grown cells of glycolate oxidase, the first enzyme in the pathway. Glycolaldehyde dehydrogenase was partially characterized as an enzyme of this new metabolic pathway in E. coli, and glycolate was identified as the product of the reaction. This enzyme used NAD and NADP as coenzymes, although the NADP-dependent activity was about 10 times lower than the NAD-dependent activity. Uptake of [14C]ethylene glycol was dependent on the presence of the enzymes capable of metabolism of ethylene glycol. Glycolaldehyde and glycolate were identified as intermediate metabolites in the pathway.     

Mitochondrial aldehyde dehydrogenase from higher plants

Aldehyde dehydrogenase has been purified to homogeneity from mitochondria of potato tubers and pea epicotyls. Although the enzyme had a high affinity for glycolaldehyde it also had a high affinity for a number of other aliphatic and arylaldehydes. It is proposed that the codification glycolaldehyde dehydrogenase (EC 1.2.1.22) should be abandoned in favour of mitochondrial aldehyde dehydrogenase (EC 1.2.1.3). The purified enzyme showed esterase activity and had properties similar to those reported for the mammalian mitochondrial aldehyde dehydrogenase. Although the natural substrate(s) for the enzyme is not known, the kinetic properties of the enzyme are consistent with it playing a role in the oxidation of acetaldehyde, glycolaldehyde and indoleacetaldehyde.     

Purification of NAD-dependent mannitol dehydrogenase from celery suspension cultures.

Mannitol dehydrogenase, a mannitol:mannose 1-oxidoreductase, constitutes the first enzymatic step in the catabolism of mannitol in nonphotosynthetic tissues of celery (Apium graveolens L.). Endogenous regulation on the enzyme activity in response to environmental cues is critical in modulating tissue concentration of mannitol, which, importantly, contribute to stress tolerance of celery. The enzyme was purified to homogeneity from celery suspension cultures grown on D-mannitol as the carbon source. Mannitol dehydrogenase was purified 589-fold to a specific activity of 365 mumol h-1 mg-1 protein with a 37% yield of enzyme activity present in the crude extract. A highly efficient and simple purification protocol was developed involving polyethylene glycol fractionation, diethylaminoethyl-anion-exchange chromatography, and NAD-agarose affinity chromatography using NAD gradient elution. Sodium dodecylsulfate gel electrophoresis of the final preparation revealed a single 40-kD protein. The molecular mass of the native protein was determined to be approximately 43 kD, indicating that the enzyme is a monomer. Polyclonal antibodies raised against the enzyme inhibited enzymatic activity of purified mannitol dehydrogenase. Immunoblots of crude protein extracts from mannitol-grown celery cells and sink tissues of celery, celeriac, and parsley subjected to sodium dodecyl sulfate gel electrophoresis showed a single major immuno-reactive 40-kD protein.     

Disruption of the fucose pathway as a consequence of genetic adaptation to propanediol as a carbon source in Escherichia coli.

In Escherichia coli, L-fucose is dissimilated via an inducible pathway mediated by L-fucose permease, L-fucose isomerase, L-fucose kinase, and L-fuculose 1-phosphate aldolase. The last enzyme cleaves the six-carbon substrate into dihydroxyacetone phosphate and L-lactaldehyde. Aerobically, lactaldehyde is oxidized to L-lactate by a nicotinamide adenine dinucleotide (NAD)-linked dehydrogenase. Anaerobically, lactaldehyde is reduced by an NADH-COUPLED REDUCTASE TO L-1,2-propanediol, which is lost into the medium irretrievably, even when oxygen is subsequently introduced. Propanediol excretion is thus the end result of a dismutation that permits further anaerobic metabolism of dihydroxy-acetone phosphate. A mutant selected for its ability to grow aerobically on propanediol as a carbon and energy source was reported to produce lactaldehyde reductase constitutively and at high levels, even aerobically. Under the new situation, this enzyme serves as a propanediol dehydrogenase. It was also reported that the mutant had lost the ability to grow on fucose. In the present study, it is shown that in wild-type cells the full synthesis of lactaldehyde dehydrogenase requires the presence of both molecular oxygen and a small molecule effector, and the full synthesis of lactaldehyde reductase requires anaerobiosis and the presence of a small molecule effector. The failure of mutant cells to grow on fucose reflects the impairment of a regulatory element in the fucose system that prevents the induction of the permease, the isomerase, and the kinase. The aldolase, on the other hand, is constitutively synthesized. Three independent fucose-utilizing revertants of the mutant all produce the permease, the isomerase, the kinase, as well as the aldolase, constitutively. These strains grow less well than the parental mutant on propanediol.     

Purification of the 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes of Neurospora crassa mitochondria

A simple purification procedure for the 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase complexes of Neurospora crassa mitochondria is described. After fractionated precipitations with polyethylene glycol, elimination of thiol proteins, and gel-filtration chromatography, the resulting preparations contained both activities. Covalent chromatography on thiol-activated Sepharose CL-4B allowed the specific binding of the 2-oxoglutarate dehydrogenase complex activity in the presence of 2-oxoglutarate, whereas the pyruvate dehydrogenase complex activity was retained in the presence of pyruvate. The purified 2-oxoglutarate dehydrogenase complex showed 4 protein bands by electrophoresis under dissociating conditions with apparent molecular weights of 160,000, 56,200, 55,600, 52,600 and a Km value of 3.8 X 10(-4) M for 2-oxoglutarate. The purified pyruvate dehydrogenase complex showed 5 protein bands with apparent molecular weights of 160,000, 57,600, 55,600, 52,500 and 37,100 and a Km value of 3.2 X 10(-4) M for pyruvate.     

The genetic control of molybdoflavoproteins in Aspergillus nidulans. II. Use of NADH dehydrogenase activity associated with xanthine dehydrogenase to investigate substrate and product inductions

Summary An NADH dehydrogenase activity is induced together with xanthine dehydrogenase I in Aspergillus nidulans wild type strains. The two activities have the same mobility in polyacrylamide gels (Fig.1) and are immunologically indistinguishable (Fig.2). Several hxB mutants which lack xanthine dehydrogenase activity but conserve the associated NADH dehydrogenase activity were used to determine that uric acid, but not hypoxanthine, is an inducer of the enzyme (Figs. 3 and 4). This fact together with results reported previously (Scazzocchio and Darlington, 1968) indicate that the induction of xanthine dehydrogenase I and urate oxidase requires the product specified by the uaY gene, as well as the common effector, urie acid.Paper I of this series is Scazzocchio, Holl and Foguelman (1973).     

Bifunctional protein in carrot contains both aspartokinase and homoserine dehydrogenase activities

We have purified homoserine dehydrogenase to homogeneity and subjected polypeptide fragments derived from digests of the protein to amino acid sequencing. The amino acid sequence of homoserine dehydrogenase from carrot (Daucus carota) indicates that in carrot both aspartokinase and homoserine dehydrogenase activities reside on the same protein. Additional evidence that aspartokinase and homoserine dehydrogenase reside on a bifunctional protein is provided by coelution of activities during purification steps and by enzyme-specific gel staining techniques. Highly purified fractions containing aspartokinase activity were stained for aspartokinase activity, homoserine dehydrogenase activity, and protein. These gels confirmed that aspartokinase activity and homoserine dehydrogenase activity were present on the same protein. This arrangement of aspartokinase and homoserine dehydrogenase activities residing on the same protein is also found in Escherichia coli, which has two bifunctional enzymes, aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II. The amino acid sequence of the major form of homoserine dehydrogenase from carrot cell suspension cultures most closely resembles that of the E. coli ThrA gene product aspartokinase I-homoserine dehydrogenase I.     

Purification of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase by gel filtration chromatography.

A procedure is presented for the large-scale purification of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12). Sephadex G-100 chromatography was more effective than repeated recrystallizations for removing heme impurities, hence these proteins appear to cocrystallize. The column purified enzyme has full enzymatic activity according to dehydrogenase, esterase, and acetyl phosphatase assays.     

Identity of Escherichia coli D-1-amino-2-propanol:NAD+ oxidoreductase with E. coli glycerol dehydrogenase but not with Neisseria gonorrhoeae 1,2-propanediol:NAD+ oxidoreductase.

The properties of D-1-amino-2-propanol oxidoreductase from wild-type Escherichia coli have been compared with those of a glycerol dehydrogenase from mutant E. coli 424 and of a 1,2-propanediol oxidoreductase from Neisseria gonorrhoeae. Several independent lines of evidence indicate that the former two enzymes are identical. (i) Both enzymatic activities purified to virtual homogeneity in an identical manner, and the ratio of specific activities (glycerol/aminopropanol) remained constant at all stages. (ii) When electrophoresed, both purified enzymes showed a major as well as a minor band of protein coincident with activity, and these two bands from each enzyme had the same mobility. (iii) The subunit molecular weights and isoelectric points were identical for each enzyme, and (iv) kinetic constants (Km and Vmax values) determined with three different substrates were the same. The somewhat greater stability of the glycerol dehydrogenase to controlled heat denaturation at 74 degrees C was the only difference observed between these two enzymes. In contrast, D-1-amino-2-propanol oxidoreductase was found to be immunochemically and kinetically distinct from the 1,2-propanediol oxidoreductase from N. gonorrhoeae.     

Purification and properties of the NAD+-dependent (type D) and O2-dependent (type O) forms of rat liver xanthine dehydrogenase.

The xanthine-oxidizing enzyme of rat liver has been purified as an NAD⁺-dependent dehydrogenase (type D) and as the O₂-dependent oxidase (type O). The purified D and O variants are nearly homogenous as judged by polyacrylamide discontinuous gel electrophoresis and are indistinguishable on sodium dodecyl sulfate-urea gels. The absorption spectrum of the type D enzyme is indistinguishable from that of the type O enzyme and closely resembles the spectra of xanthine-oxidizing enzymes from other sources. The types D and O enzymes have essentially the same cofactor composition. Oxidation of xanthine by type D is stimulated by NAD⁺ with concomitant NADH formation. Type D is able to utilize NADH as well as xanthine as electron donor to various acceptors, in contrast to type O that is unable to oxidize NADH. Arsenite, cyanide and methanol completely abolish xanthine oxidation by the type D enzyme while affecting the activities with NADH to varying extents. In these respects rat liver xanthine dehydrogenase closely resembles chicken liver xanthine dehydrogenase. However, in contrast to the avian enzyme, the purified rat liver enzyme is unstable as a dehydrogenase and is gradually converted to an oxidase. This conversion is accompanied by an increase in the aerobic xanthine → cytochrome c activity. The native type D enzyme in rat liver extracts is precipitable with antibody prepared against purified type O. The K_m for xanthine is not significantly different for the two forms.     

Purification and properties of Klebsiella aerogenes D-arabitol dehydrogenase.

An Escherichia coli K12 strain was constructed that synthesized elevated quantities of Klebsiella aerogenes D-arabitol dehydrogenase; the enzyme accounted for about 5% of the soluble protein in this strain. Some 280 mg of enzyme was purified from 180 g of cell paste. The purified enzyme was active as a monomer of 46,000 mol.wt. The amino acid composition and kinetic constants of the enzyme for D-arabitol and D-mannitol are reported. The apparent Km for D-mannitol was more than 3-fold that for D-arabitol, whereas the maximum velocities with both substrates were indistinguishable. The enzyme purified from the E. coli K12 construct was indistinguishable by the criteria of molecular weight, electrophoretic mobility in native polyacrylamide gel and D-mannitol/D-arabitol activity ratio from D-arabitol dehydrogenase synthesized in wild-type K. aerogenes. Purified D-arabitol dehydrogenase showed no immunological cross-reaction with K. aerogenes ribitol dehydrogenase. During electrophoresis in native polyacrylamide gels, oxidation by persulphate catalysed the formation of inactive polymeric forms of the enzyme. Dithiothreitol and pre-electrophoresis protected against this polymerization.     

Purification and site-specific immobilization of genetically engineered glucose dehydrogenase on thiopropyl-sepharose

The gene encoding glucose dehydrogenase (EC 1.1.1.47) from Bacillus subtilis was inserted in a plasmid 1.0 kb downstream from a lac promoter, resulting in a 70-fold higher production of the enzyme when expressed in Escherichia coli. A glucose dehydrogenase mutant containing a cysteine residue at position 44 could also be expressed at the same high level. This single cysteine residue was used as an ‘affinity tag’ to simplify the purification procedure as well as for site-specific immobilization of glucose dehydrogenase on Thiopropyl-Sepharose. This enzyme was purified to homogeneity with a final recovery of 65% and a specific activity of 240 U/mg. The oriented immobilization resulted in increased thermal stability.     

Purification and characterization of 7beta-hydroxysteroid dehydrogenase from rabbit liver microsomes

7beta-Hydroxysteroid dehydrogenase (7beta-HSD), a specific enzyme active in the metabolization of 7beta-hydroxycholesterol, was purified about 300-fold from male rabbit liver microsomes using ion exchange, hydroxylapatite, 2'5'ADP Sepharose 4B, and high-performance liquid chromatography on the basis of its catalytic activity. The specific activity of the purified enzyme was 276 nmol/min/mg protein. The molecular weight of the purified enzyme was 34,000. The preferred coenzyme was beta-NADP+. The optimum pH for oxidation was around 7.7 in potassium phosphate buffer, and 11.0 in glycine-NaOH buffer. The purified enzyme catalyzed the synthesis of not only 7beta-hydroxycholesterol but also corticosterone and hydrocortisone. Enzyme activities toward these three substrates accompanied all purification steps of 7beta-HSD. The amino acid sequence of the N-terminal of the purified enzyme showed that 7beta-HSD had sequence similarity to rabbit type I 11beta-hydroxysteroid dehydrogenase (11beta-HSD), indicating that 7beta-HSD may belong to the rabbit type I 11beta-HSD family and may play the same role in the metabolism of 11-hydroxysteroids and 7-hydroxysterols.     

Expression and purification of His-tagged rat mitochondrial medium-chain acyl-CoA dehydrogenase wild-type and Arg256 mutant proteins

Mitochondrial medium-chain acyl-CoA dehydrogenase is a key enzyme for the beta-oxidation of fatty acids, and the deficiency of this enzyme in patient has been previously reported. We cloned the gene of rat mitochondrial medium-chain acyl-CoA dehydrogenase into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 3' of the gene. The cloned gene was overexpressed in Escherichia coli and the soluble protein was purified with a nickel Hi-Trap chelating metal affinity column in 88% yield to apparent homogeneity. The specific activity of the purified His-tagged rat mitochondrial medium-chain acyl-CoA dehydrogenase was 4.0 U/mg. Arg256 is a highly conserved amino acid, which may play an important role in enzymatic reaction based on the crystal structure of medium-chain acyl-CoA dehydrogenase. We constructed four mutant expression plasmids of the enzyme using site-directed mutagenesis. Mutant proteins were overexpressed in E. coli and purified with a nickel metal affinity column. Kinetic studies of wild-type and mutant proteins were carried out, and the result confirmed that Arg256 is a very important residue of rat mitochondrial medium-chain acyl-CoA dehydrogenase. Our overexpression in E. coli and one-step purification of the highly active rat mitochondrial medium-chain acyl-CoA dehydrogenase greatly facilitated our further investigation of this enzyme, and our result from site-directed mutagenesis increased our understanding of medium-chain acyl-CoA dehydrogenase.     

Messenger RNA guanylyltransferase from Saccharomyces cerevisiae. Large scale purification, subunit functions, and subcellular localization

Messenger RNA capping enzyme (GTP:mRNA guanylyltransferase) purified from yeast Saccharomyces cerevisiae consisted of two polypeptides (45 and 39 kDa) and possessed two enzymatic activities, i.e. mRNA guanylyltransferase and RNA 5'-triphosphatase (Itoh, N., Mizumoto, K., and Kaziro, Y. (1984) J. Biol. Chem. 259, 13923-13929). In this paper, we describe an improved procedure suitable for the large scale purification of the enzyme. The steps include glass beads disruption of the cells and several ion-exchange and affinity column chromatographies. The enzyme was purified from kilogram quantities of yeast cells to apparent homogeneity. The purified enzyme had an approximate Mr of 180,000 and consisted of two heterosubunits of 80 and 52 kDa and had the same two enzymatic activities as above. We consider that this is the more intact form of the enzyme. Using the in situ assays on sodium dodecyl sulfate-polyacrylamide gels, RNA 5'-triphosphatase, and mRNA guanylyltransferase activities were located on the 80- and 52-kDa chains, respectively. In agreement with this, the 52-kDa enzyme-[32P]GMP complex was formed on incubation of the enzyme with [alpha-32P]GTP. Guinea pig antisera against purified yeast capping enzyme recognized both 80- and 52-kDa chains in Western blot analysis. The antibody did not cross-react with the enzymes from rat liver. Artemia salina, or vaccinia virus. Nuclear localization of the enzyme was demonstrated by immunofluorescence microscopy.     

UGA nonsense mutation in the alcohol dehydrogenase gene of Drosophila melanogaster

A mutant gene, which we have designated AdhnB, codes for a defective form of the enzyme alcohol dehydrogenase in Drosophila melanogaster. We show that the polypeptide encoded by AdhnB is approximately 2000 Mr smaller than the protein synthesized under the direction of the wild-type alcohol dehydrogenase gene. In contrast, the alcohol dehydrogenase mRNA produced by both genes is the same size. We cloned and sequenced a portion of the protein-coding region of AdhnB and compared it to the same region in the wild-type gene. We found a single base substitution: a change of the TGG tryptophan codon at amino acid 235 to a TGA termination codon. This nonsense mutation accounts for the observed reduction in size of the alcohol dehydrogenase polypeptide. In further studies, we found that the steady-state levels of alcohol dehydrogenase mRNA in flies carrying the AdhnB gene and the wild-type alcohol dehydrogenase gene were indistinguishable. However, the steady-state level of alcohol dehydrogenase polypeptide was reduced to 1% of wild-type levels in flies with the AdhnB gene. Moreover, the rate of alcohol dehydrogenase synthesis in mutant flies was reduced to 50% of that found in wild type. The aberration in AdhnB thus affects both the rate of synthesis and the rate of degradation of the alcohol dehydrogenase peptide. AdhnB is the first reported nonsense mutant in Drosophila.     

L-lyxose metabolism employs the L-rhamnose pathway in mutant cells of Escherichia coli adapted to grow on L-lyxose.

Escherichia coli cannot grow on L-lyxose, a pentose analog of the 6-deoxyhexose L-rhamnose, which supports the growth of this and other enteric bacteria. L-Rhamnose is metabolized in E. coli by a system that consists of a rhamnose permease, rhamnose isomerase, rhamnulose kinase, and rhamnulose-1-phosphate aldolase, which yields the degradation products dihydroxyacetone phosphate and L-lactaldehyde. This aldehyde is oxidized to L-lactate by lactaldehyde dehydrogenase. All enzymes of the rhamnose system were found to be inducible not only by L-rhamnose but also by L-lyxose. L-Lyxose competed with L-rhamnose for the rhamnose transport system, and purified rhamnose isomerase catalyzed the conversion of L-lyxose into L-xylulose. However, rhamnulose kinase did not phosphorylate L-xylulose sufficiently to support the growth of wild-type E. coli on L-lyxose. Mutants able to grow on L-lyxose were analyzed and found to have a mutated rhamnulose kinase which phosphorylated L-xylulose as efficiently as the wild-type enzyme phosphorylated L-rhamnulose. Thus, the mutated kinase, mapped in the rha locus, enabled the growth of the mutant cells on L-lyxose. The glycolaldehyde generated in the cleavage of L-xylulose 1-phosphate by the rhamnulose-1-phosphate aldolase was oxidized by lactaldehyde dehydrogenase to glycolate, a compound normally utilized by E. coli.     

Purification of the putA gene product. A bifunctional membrane-bound protein from Salmonella typhimurium responsible for the two-step oxidation of proline to glutamate

In this paper we report the purification of a protein which is able to catalyze both the proline oxidase and the pyrroline-5-carboxylic acid dehydrogenase activities necessary for the oxidation of proline to glutamic acid. The purification involves the preparation of a crude membrane pellet, detergent solubilization, ammonium sulfate fractionation, and DEAE-chromatography. We are able to obtain an essentially pure preparation (greater than 95% pure) after only a 52-fold purification, demonstrating that the protein is a major protein in cells fully induced for proline utilization. Both proline oxidase and pyrroline-5-carboxylic acid dehydrogenase activities co-purity throughout our purification. Velocity sedimentation of the purified protein demonstrates that both proline oxidase and pyrroline-5-carboxylic acid dehydrogenase activities co-sediment. Early in the purification procedure we are able to detect two species of protein which have both proline oxidase and pyrroline-5-carboxylic acid dehydrogenase activities. Our procedure purifies only the larger molecular weight species. The purified protein is a dimer composed of identical 132,000-dalton subunits. Analysis of mutants defective for proline utilization demonstrate that the bifunctional enzyme is the putA gene product.