Localisation of the glucose-fructose oxidoreductase in wild type and overproducing strains of Zymomonas mobilis

Abstract The enzyme glucose-fructose oxidoreductase (GFOR) from the Gram-negative ethanologenic bacterium Zymomonas mobilis was purified to homogeneity and was shown to be a tetrameric protein with a subunit size of M _r 42 500. Using immunogold-labelling in combination with electron microscopy, ultrathin sections of Z. mobilis wild type cells showed that the enzyme GFOR is located in the periplasm off the bacterial cells. Z. mobilis strains which carried the cloned gfo gene on plasmid pSUP104, had 5–6-fold increased GFOR enzyme activities. Moreover, these cells accumulated large amounts of a presumable unprocessed pre-GFOR protein ( M _r 48 000).     

Photoreduction of monovinyl- and divinyl-protochlorophyllide in vitro and in vivo in the light-dependent greening mutant C-2A' of Scenedesmus obliquus

Dark-grown cells of mutant C-2A' of Scenedesmus obliquus accumulate monovinyl-and mainly divinyl-protochlorophyllide (PChlide). Both PChlide-forms are equally well photoconverted in vitro by the NADPH-protochlorophyllide oxidoreductase of the light-dependent greening mutant C-2A'of Scenedesmus obliquus. The chlorophyllide (Chlide) resulting from this photoconversion in vivo has a predominantly monovinyl character. Only small traces of a transient Chlide-form with divinyl fluorescence could be detected.     

How inter-subunit contacts in the membrane domain of complex I affect proton transfer energetics

The respiratory complex I is a redox-driven proton pump that employs the free energy released from quinone reduction to pump protons across its complete ca. 200?Å wide membrane domain. Despite recently resolved structures and molecular simulations, the exact mechanism for the proton transport process remains unclear. Here we combine large-scale molecular simulations with quantum chemical density functional theory (DFT) models to study how contacts between neighboring antiporter-like subunits in the membrane domain of complex I affect the proton transfer energetics. Our combined results suggest that opening of conserved Lys/Glu ion pairs within each antiporter-like subunit modulates the barrier for the lateral proton transfer reactions. Our work provides a mechanistic suggestion for key coupling effects in the long-range force propagation process of complex I.     

Liver aldehyde oxidase and xanthine oxidase genetics in the mouse

A 'null' activity variant for the major liver isozyme of aldehyde oxidase (AOX-1) in adult male mice and an electrophoretically distinct, high activity variant of the second liver isozyme (AOX-2) were used to examine the segregation of the genetic loci encoding these enzymes (Aox-1 and Aox-2 respectively) in breeding studies. A single recombinant between these loci was observed among the 147 backcross progeny examined, which confirms a previous report (Holmes, 1979) for close linkage and genetic distinctness of the two loci. An activity variant for mouse liver xanthine oxidase (XOX) is also reported which behaved as though controlled by codominant alleles at a single locus (designated Xox-1 ). Genetic analyses showed that the Xox-1 locus segregated independently of the multiple- A ox loci.     

Activité oxydasique de l''oxydoréductase de la xanthine, spécifique de la classe des mammifères

In every species other than mammals xanthine oxidoreductase behaves as a dehydrogenase, never as an oxidase. In three mammalian species, the enzyme acts intracellularly as a dehydrogenase, but its class-specific ambivalence allows its extracellular conversion into an oxidase.

Résumé

En dehors de la classe des Mammifères, l'oxydoréductase de la xanthine ne se comporte jamais comme une oxydase. Chez les Mammifères, une ambivalence de la molécule permet l'expression d'une activité déshydrogénasique (NAD) dans la cellule, et d'une activité oxydasique en dehors de la cellule.     

Transformation of photoinactive to photoactive protochlorophyllide in isolated prolamellar bodies of wheat (Triticum aestivum) exposed to low pH and ATP

Isolated prolamellar bodies from the etioplasts of dark-grown wheat ( Triticum aestivum L. cv. Walde, Weibull) contain the enzyme NADPH-protochlorophyllide oxidoreductase. The organisation of this enzyme in a pigment-protein complex results in fluorescence emission maxima at 633 and 657 nm. Isolated prolamellar bodies stored in darkness for 24 or 48 h at 4°C (pH 7.2) in the presence of NADPH showed a fluorescence emission ratio 657/633 nm around 4 at −196°C. With acidic conditions this fluorescence ratio increased, with an optimum at pH 5.5. Such an increase was even more pronounced in the presence of ATP and NADPH with ratios up to 8, but was completely blocked when the sulfhydryl inhibitor, dithiobis-nitrobenzoic acid, was added. As shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis the amount of NADPH-protochlorophyllide oxidoreductase in the prolamellar bodies did not change during storage for 24 or 48 h.
The total amount of protochlorophyllide measured in acetone extracts did not change significantly during storage for 48 h. The values were similar for storage at pH 7.2 and 5.5, but at lower pH (around 5) the pigment content decreased to a third.
The most plausible explanation for the increase in fluorescence ratio is that low pH and ATP give rise to a change in conformation, which results in transformation of the short wavelength (633 nm) fluorescing protochlorophyllide to the long wavelength (657 nm) fluorescing form.     

Anaerobic metabolism of l-phenylalanine via benzoyl-CoA in the denitrifying bacterium Thauera aromatica

The anaerobic metabolism of phenylalanine was studied in the denitrifying bacterium Thauera aromatica, a member of the β-subclass of the Proteobacteria. Phenylalanine was completely oxidized and served as the sole source of cell carbon. Evidence is presented that degradation proceeds via benzoyl-CoA as the central aromatic intermediate; the aromatic ring-reducing enzyme benzoyl-CoA reductase was present in cells grown on phenylalanine. Intermediates in phenylalanine oxidation to benzoyl-CoA were phenylpyruvate, phenylacetaldehyde, phenylacetate, phenylacetyl-CoA, and phenylglyoxylate. The required enzymes were detected in extracts of cells grown with phenylalanine and nitrate. Oxidation of phenylalanine to benzoyl-CoA was catalyzed by phenylalanine transaminase, phenylpyruvate decarboxylase, phenylacetaldehyde dehydrogenase (NAD⁺), phenylacetate-CoA ligase (AMP-forming), enzyme(s) oxidizing phenylacetyl-CoA to phenylglyoxylate with nitrate, and phenylglyoxylate:acceptor oxidoreductase. The capacity for phenylalanine oxidation to phenylacetate was induced during growth with phenylalanine. Evidence is provided that α-oxidation of phenylacetyl-CoA is catalyzed by a membrane-bound enzyme. This is the first report on the complete anaerobic degradation of an aromatic amino acid and the regulation of this process. Received: 6 March 1997 / Accepted: 16 May 1997     

Isolation and characterization of photoactive complexes of NADPH:protochlorophyllide oxidoreductase from wheat

A photoactive substrate-enzyme complex of the NADPH:protochlorophyllide oxidoreductase (POR; EC 1. 3. 1. 33) was purified from etiolated Triticum aestivum L. by gel chromatography after solubilization of prolamellar bodies by dodecyl-maltoside. Irradiation by a 1-ms flash induced the phototransformation of protocholorophyllide a (Pchlide) with −196 °C absorbance and emission maxima at 640 and 643 nm, respectively. The apparent molecular weight of this complex was 112 ± 24 kDa, which indicates aggregation of enzyme subunits. By lowering the detergent concentration in the elution buffer, a 1080 ± 250-kDa particle was obtained which displayed the spectral properties of the predominant form of photoactive Pchlide in vivo (−196 °C absorbance and fluorescence maxima at 650 and 653 nm). In this complex, POR was the dominant polypeptide. Gel chromatography in the same conditions of an irradiated sample of solubilized prolamellar bodies indicated rapid disaggregation of the complex after Pchlide phototransformation. High performance liquid chromatographic analysis of the POR complexes obtained using two detergent concentrations indicates a possible association of zeaxanthin and violaxanthin with the photoactive complex. Received: 25 February 1998 / Accepted: 8 June 1998