Purification and characterization of D(+)-carnitine dehydrogenase from Agrobacterium sp.--a new enzyme of carnitine metabolism.

D(+)-Carnitine dehydrogenase from Agrobacterium sp. catalyzes the oxidation of D(+)-carnitine to 3-dehydrocarnitine as initial step of D(+)-carnitine degradation. The NAD(+)-specific, cytosolic enzyme was purified 126-fold to apparent electrophoretic homogeneity by 4 chromatographic steps. The molecular mass of the native enzyme was estimated to be 88 kDa by size-exclusion chromatography. It seems to be composed of 3 identical subunits with a relative molecular mass of 28 kDa as found by sodium dodecyl sulfate polyacrylamide gel electrophoresis and laser-induced mass spectrometry. The isoelectric point was found to be 4.7-5.0. The optimum temperature is 37 degrees C and the optimum pH for the oxidation and the reduction reaction are 9.0-9.5 and 5.5-6.5, respectively. The purified enzyme was further characterized with respect to substrate specificity, kinetic parameters and amino terminal sequence. Analogues of D(+)-carnitine (L(-)-carnitine, crotonobetaine, gamma-butyrobetaine, carnitine amide, glycine betaine, choline) are competitive inhibitors of D(+)-carnitine oxidation. The equilibrium constant of the reaction of D(+)-carnitine dehydrogenase was determined to be 2.2 x 10(-12). The purified D(+)-carnitine dehydrogenase has similar kinetic properties to the L(-)-carnitine dehydrogenase from the same microorganism as well as to L(-)-carnitine dehydrogenases of other bacteria.

Purification and properties of carnitine dehydratase from Escherichia coli--a new enzyme of carnitine metabolization.

Carnitine dehydratase from Escherichia coli 044 K74 is an inducible enzyme detectable in cells grown anaerobically in the presence of L(-)-carnitine or crotonobetaine. It has been purified 500-fold to electrophoretic homogeneity by chromatography on phenyl-Sepharose, hydroxyapatite, DEAE-Sepharose, second phenyl-Sepharose and finally gel filtration on a Sephadex G-100 column. During the purification procedure a low-molecular-weight effector essential for enzyme activity was separated from the enzyme. The addition of this still unknown effector caused reactivation of the apoenzyme. The relative molecular mass of the apoenzyme has been estimated to be 85,000. It seems to be composed of two identical subunits with a relative molecular mass of 45,000. The purified and reactivated enzyme has been further characterized with respect to pH and temperature optimum (7.8 and 37-42 degrees C), equilibrium constant (Keq = 1.5 +/- 0.2) and substrate specifity. The enzyme is inhibited by thiol reagents. The Km value for crotonobetaine is 1.2.10(-2) M. gamma-Butyrobetaine, D(+)-carnitine and choline are competitive inhibitors of crotonobetaine hydration.

Purification and properties of L(-)-carnitine dehydrogenase from Agrobacterium sp.

L(-)-Carnitine:NAD+ oxidoreductase, EC 1.1.1.108, from Agrobacterium sp. catalyzes the oxidation of L(-)-carnitine to 3-dehydrocarnitine as initial step of L(-)-carnitine degradation. The enzyme was purified 76-fold by four chromatographic steps. A high substrate specificity for L(-)-carnitine and NAD+ was observed. The molecular mass of the native enzyme is 114 kDa and it consists of two identical subunits as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point was found to be 5.2-5.4. The optimum temperature is 45 degrees C and the optimum pH for the oxidation and the reduction reaction are 9.5 and 5.5-6.5, respectively. Kinetic parameters and amino-terminal sequence were determined. The oxidation reaction is inhibited by D(+)-carnitine, trimethylamine, several metal ions and cetyltrimethylammoniumbromide (CTAB).

Crotonobetaine reductase from Escherichia coli consists of two proteins.

Crotonobetaine reductase from Escherichia coli is composed of two proteins (component I (CI) and component II (CII)). CI has been purified to electrophoretic homogeneity from a cell-free extract of E. coli O44 K74. The purified protein shows l(-)-carnitine dehydratase activity and its N-terminal amino acid sequence is identical to the caiB gene product from E. coli O44 K74. The relative molecular mass of CI has been determined to be 86100. It is composed of two identical subunits with a molecular mass of 42600. The isoelectric point of CI was found to be 4.3. CII was purified from an overexpression strain in one step by ion exchange chromatography on Fractogel EMD TMAE 650(S). The N-terminal amino acid sequence of CII shows absolute identity with the N-terminal sequence of the caiA gene product, i.e. of the postulated crotonobetaine reductase. The relative molecular mass of the protein is 164400 and it is composed of four identical subunits of molecular mass 41500. The isoelectric point of CII is 5.6. CII contains non-covalently bound FAD in a molar ratio of 1:1. In the crotonobetaine reductase reaction one dimer of CI associates with one tetramer of CII. A still unknown low-molecular-mass effector described for the l(-)-carnitine dehydratase is also necessary for crotonobetaine reductase activity. Monoclonal antibodies were raised against the two components of crotonobetaine reductase.

Bacterial carnitine metabolism.

L-(-)-Carnitine is a ubiquitously occurring substance, essential for the transport of long-chain fatty acids through the inner mitochondrial membrane. Bacteria are able to metabolize this trimethylammonium compound in three different ways. Some, especially Pseudomonas species, assimilate L-(-)-carnitine as sole source of carbon and nitrogen. The first catabolic step is catalysed by the L-(-)-carnitine dehydrogenase. Others, for instance, Acinetobacter species, degrade only the carbon backbone, with formation of trimethylamine. Finally, various members of the Enterobacteriaceae are able to convert carnitine, via crotonobetaine, to gamma-butyrobetaine in the presence of C and N sources and under anaerobic conditions. This two-step pathway, including a L-(-)-carnitine dehydratase and the crotonobetaine reductase, was demonstrated in Escherichia coli. The DNA sequence encompassing the cai genes of E. coli, which encode the carnitine pathway, has been determined. Some bacteria are also able to metabolize the non-physiological D-(+)-carnitine, which results as a waste product in some chemical procedures for L-(-)-carnitine production based on the resolution of racemic carnitine.

Epigenetic regulation of carnitine metabolising enzymes in Proteus sp. under aerobic conditions.

Proteus sp. is able to catalyse the reversible transformation of crotonobetaine into L(-)-carnitine during aerobic growth. Contrary to other Enterobacteriaceae no reduction of crotonobetaine into gamma-butyrobetaine could be detected in the culture supernatants. Activities of L(-)-carnitine dehydratase, carnitine racemasing system and crotonobetaine reductase could be determined enzymatically in cell-free extracts of Proteus sp. Small amounts of gamma-butyrobetaine were found in cell-free extracts, indicating that it accumulates in the cell and inhibits the crotonobetaine reductase. Crotonobetaine and L(-)-carnitine were able to induce enzymes of carnitine metabolism. gamma-Butyrobetaine and glucose repress carnitine metabolism in Proteus sp. Other betaines are neither inducers nor repressors. Monoclonal antibodies against purified CaiA from Escherichia coli O44K74 recognise an analogous protein in cell-free extract of Proteus sp. No cross-reactivity could be detected with monoclonal antibodies against purified CaiB and CaiD from E. coli O44K74.

Metabolism of L(-)-carnitine by Enterobacteriaceae under aerobic conditions.

Different Enterobacteriaceae, such as Escherichia coli, Proteus vulgaris and Proteus mirabilis, are able to convert L(-)-carnitine, via crotonobetaine, into gamma-butyrobetaine in the presence of carbon and nitrogen sources under aerobic conditions. Intermediates of L(-)-carnitine metabolism (crotonobetaine, gamma-butyrobetaine) could be detected by thin-layer chromatography. In parallel, L(-)-carnitine dehydratase, carnitine racemasing system and crotonobetaine reductase activities were determined enzymatically. Monoclonal antibodies against purified CaiB and CaiA from E. coli O44K74 were used to screen cell-free extracts of different Enterobacteriaceae (E. coli ATCC 25922, P. vulgaris, P. mirabilis, Citrobacter freundii, Enterobacter cloacae and Klebsiella pneumoniae) grown under aerobic conditions in the presence of L(-)-carnitine.

Metabolism of D: (+)-carnitine by Escherichia coli.

Escherichia coli 044 K74 grown under anaerobic conditions in the presence of L: (-)-carnitine is able to convert D: (+)-carnitine into the L: (-)-enantiomer. This activity is repressed by electron acceptors such as oxygen and nitrate as well as by glucose. D: (+)-Carnitine shows no effect on the induction or repression of the corresponding enzyme or enzyme system. Resting cells of E. coli 044 K74 were used for the formation of L: (-)-carnitine from D: (+)-carnitine. The maximum obtained yield was 50%. γ-Butyrobetaine was formed as a by-product.

[Inhibiton of isocitrate dehydrogenase and isocitrate lyase from Acinetobacter calcoaceticus by acids of the citrate and glyoxylate cycle]. / Die Hemmung der Isozitratdehydrogenase und der Isozitratlyase aus Acinetobacter calcoaceticus durch Säuren des Zitrat- und Glyoxylatzyklus

Acinetobacter calcoaceticus contains two forms of NADP+-dependent isocitrate dehydrogenases differing, among others, by their molecular weights and regulatory properties. The regulation of the high-molecular form of isocitrate dehydrogenase and of isocitrate lyase by organic acids, either belonging or related to the citrate and glyoxalate cycle, is investigated. While alpha-ketoglutarate and oxalacetate competitively inhibit the isocitrate dehydrogenase against Ds-isocitrate, glyoxylate and pyruvate were found to increase Vmax and to lower the KM value for Ds-isocitrate and NADP+. Simultaneous addition of oxalacetate and glyoxylate (not, however, addition of the nonenzymatically formed condensation product of both compound) nullified the activation of isocitrate dehydrogenase by glyoxylate, and potentiates the inhibitory effect of oxalacetate. Alpha-ketoglutarate, succinate, and phosphoenolpyruvate inhibit the isocitrate lyase in a noncompetitive fashion against DS-isocitrate; L-malate, oxalacetate and glyoxylate inhibit competitively. The intermediates of the citrate and glyoxylate cycle afford additive inhibition of the isocitrate lyase. The importance of organic acids of the citrate and glyoxylate cycle and of phosphoenolpyruvate for the regulation of the citrate and glyoxylate cycle at the level of isocitrate dehydrogenase and isocitrate lyase is discussed.

[Inhibition of the malic enzyme from Acinetobacter calcoaceticus by NADPH and NADH]. / Hemmung des Malatenzyms aus Acinetobacter calcoaceticus durch NADPH and NADH

The malic enzyme enriched from Acinetobacter calcoaceticus is inhibited by NADPH and NADH. The inhibition afforded by the reduced coenzymes is not affected by NAD+, AMP and 3'.5'-AMP. Against L-malate, NADPH inhibits the enzyme in a noncompetitive linear fashion (Ki = 1.5 x 10(-4) M), against NADP+, competitively linearly (Ki = 5.0 x 10(-5) M). While NADPH acted as a product inhibitor, NADH seems to be an allosteric effector of the malic enzyme, because with L-malate as the variable substrate in the double reciprocal plot, a nonlinear curve is obtained.

Selective and rapid solubilization of the microbial membrane enzyme aldehyde dehydrogenase.

An improved solubilization procedure for the membrane-bound quinoprotein aldehyde dehydrogenase from Acetobacter rancens CCM 1774 was established. After the first solubilization of membrane enzymes by Brij 35 which provided important extraction of membrane proteins other than aldehyde dehydrogenase, the application of Trition X-100 resulted in an almost 20-fold purification of quinoprotein aldehyde dehydrogenase. The optimal solubilization was closely connected with definite detergent/protein ratios.

[Rubredoxin reductase in crude extracts of Acinetobacter calcoaceticus in relation to carbon source and growth phase]. / Rubredoxin-Reductase in Rohextrakten von Acinetobacter calcoaceticus in Abhängigkeit von C-Quelle und Wachstumsphase.

By immunization of rabbits with discelectrophoretically purified rubredoxin-reductase from Ac. calcoaceticus an antiserum against this enzyme was prepared. The antiserum was monospecifical to a diaphoretic activity, which had a RF-value identical with the rubredoxin-reductase-RF value. It has been shown by discelectrophoresis, immunoelectrophoresis, OUCHTERLONY technique and kinetic investigations that crude extracts of bacteria grown on C16, malate, succinate or acetate contain rubredoxin-reductase. By the LAURELL technique we demonstrated that the amount of the enzyme is nearly independent of the carbon source. There are only differences during the different growth phases.

Isolation and characterization of the extracellular lipase of Acinetobacter calcoaceticus 69 V.

The extracellular lipase of Acinetobacter calcoaceticus 69 V was purified by hydrophobic interaction chromatography to homogeneity as suggested by gel electrophoretic analysis. The lipase existed as a high molecular complex of about 300 kDa, with a subunit molecular weight of 30.5 kDa being obtained by SDS-PAGE. The hydrodynamic molecular radius obtained by gel electrophoresis was 3.27 nm. The lipase had an isoelectric point of 5.5 and was stimulated by additions of deoxycholate. The activation energy for the hydrolysis of p-nitrophenyl palmitate was 39.9 kJ mol-1. Tri-, di- and monoacylglycerols were hydrolyzed. Hg2+ and p-hydroxymercuribenzoate inhibited the enzyme activity at very low concentrations. One sulfhydryl group was found per molecule of lipase.

Effects of detergents and phospholipids on the pyridine nucleotide-independent aldehyde dehydrogenase from membranes of Acetobacter rancens.

The pyridine nucleotide-independent aldehyde dehydrogenase solubilized and purified from membranes of Acetobacter rancens CCM 1774 requires the presence of detergents for activity. While several detergents could stimulate the enzyme activity the stability of the enzyme-detergent complexes was rather low. Phospholipid substitution experiments revealed the reversibility of the loss of activity caused by phospholipid removal. Enzyme-phospholipid complexes generated from a complex phospholipid fraction exhibited an increased stability. The predominant effect of phosphatidylglycerol on both enzyme activity and stability is discussed in terms of essential boundary layer phospholipids.

Purification and characterization of an extracellular beta-lactamase produced by Acinetobacter calcoaceticus.

A beta-lactamase was purified 430-fold from the culture supernatant of Acinetobacter calcoaceticus by ion exchange chromatography on CM-Sephadex and affinity chromatography on phenylboronic-acid-agarose. The purified enzyme was homogeneous as judged by SDS-PAGE, and was characterized with respect to molecular mass (38 and 41 kDa by gel filtration on Sephadex G-75 and SDS-PAGE, respectively), pH optimum (pH 7.0), temperature optimum (45 degrees C) and isoelectric point (9.3). The beta-lactamase showed mainly cephalosporinase activity. It was inhibited by cloxacillin, carbenicillin, penicillanic acid sulphone (sulbactam) and aztreonam. It was not inhibited by clavulanic acid up to a concentration of 0.25 mM. Neither EDTA nor p-chlormercuribenzoate, up to concentrations of 1 or 100 mM, respectively, affected activity. According to these characteristics, it is a typical CEP-N cephalosporinase.