Studies on the mechanism of acetate oxidation by bacteria. VI. Comparative patterns of acetate oxidation by citrate-grown and acetate-grown Aerobacter aerogenes

The data presented in this paper indicate operation of different mechanisms for acetate oxidation by A. aerogenes, depending on the carbon source used for growth. The mechanism for citrate-grown cells appears to involve a conventional citric acid cycle, whereas acetate-grown cells appear to incorporate acetate carbon more readily via a dicarboxylic acid cycle.     

Studies on the kinetic mechanism and the phosphoryl-enzyme compound of the Escherichia coli acetate kinase reaction.

The Escherichia coli acetate kinase was further examined to determine whether a phosphoryl-enzyme intermediate (or compound) was kinetically discernible or chemically essential in the catalysis of that reaction. The extent of phosphoryl-enzyme formation as monitored by gel filtration of the enzyme-phosphoryl donor substrate complex (E-P) was found to be quite variable. Isolated E-P could be reduced with sodium borohydride and the extent of formation of α-amino-δ-hydroxyvalerate paralleled the observed degree of phosphorylation of the acetate kinase. Incubation with 1 m neutral hydroxylamine of the enzyme, which had been preincubated with acetyl-P for 30–60 min, led to parallel loss of the enzyme's ability to catalyze the net reaction and the ADP ATP exchange reaction. The percentage of activity loss depended upon the concentration of acetyl-P with which the enzyme was preincubated. These observations point toward a requirement for an unmodified active site carboxyl group which may be phosphorylated during the reaction course. The theoretical basis for a new and simple alternative substrate protocol for segregating sequential and ping-pong mechanisms is presented. When applied to acetate kinase, the findings clearly rule out a ping-pong kinetic mechanism. This was also confirmed by initial rate measurements with propionyl-P and ADP, and the weight of the evidence now clearly favors a modified random substrate addition pathway as presented herein. The data do not in any way exclude E-P formation as a requirement for catalysis. Finally, the rates of cold inactivation at 0 °C and reactivation at 29 °C were determined in order to minimize the possible influence of this phenomenon on the kinetic studies.     

Propionate oxidation in Escherichia coli: evidence for operation of a methylcitrate cycle in bacteria

Escherichia coli grew in a minimal medium on propionate as the sole carbon and energy source. Initially a lag phase of 4–7 days was observed. Cells adapted to propionate still required 1–2 days before growth commenced. Incorporation of (2-¹³C), (3-¹³C) or (²H₃)propionate into alanine revealed by NMR that propionate was oxidized to pyruvate without randomisation of the carbon skeleton and excluded pathways in which the methyl group was transiently converted to a methylene group. Extracts of propionate-grown cells contained a specific enzyme that catalyses the condensation of propionyl-CoA with oxaloacetate, most probably to methylcitrate. The enzyme was purified and identified as the already-known citrate synthase II. By 2-D gel electrophoresis, the formation of a second propionate-specific enzyme with sequence similarities to isocitrate lyases was detected. The genes of both enzymes were located in a putative operon with high identities (at least 76% on the protein level) with the very recently discovered prp operon from Salmonella typhimurium. The results indicate that E. coli oxidises propionate to pyruvate via the methylcitrate cycle known from yeast. The ¹³C patterns of aspartate and glutamate are consistent with the further oxidation of pyruvate to acetyl-CoA. Oxaloacetate is predominantly generated via the glyoxylate cycle rather than by carboxylation of phosphoenolpyruvate. Received: 28 April 1997 / Accepted: 4 July 1997     

Improved conversion of fumarate to succinate by Escherichia coli strains amplified for fumarate reductase.

Two recombinant plasmid Escherichia coli strains containing amplified fumarate reductase activity converted fumarate to succinate at significantly higher rates and yields than a wild-type E. coli strain. Glucose was required for the conversion of fumarate to succinate, and in the absence of glucose or in cultures with a low cell density, malate accumulated. Two-dimensional gel electrophoretic analysis of proteins from the recombinant DNA and wild-type strains showed that increased quantities of both large and small fumarate reductase subunits were expressed in the recombinant DNA strains.     

Increased production of succinic acid in Escherichia coli by overexpression of malate dehydrogenase

Escherichia coli NZN111 is a double mutant with inactivated lactate dehydrogenase and pyruvate formate-lyase. It cannot utilize glucose anaerobically because of its unusually high intracellular NADH/NAD(+) ratio. We have now constructed a recombinant strain, E. coli NZN111/pTrc99a-mdh, which, during anaerobic fermentation, produced 4.3 g succinic acid l(-1) from 13.5 g glucose l(-1). The NADH/NAD(+) ratio decreased from 0.64 to 0.26. Furthermore, dual-phase fermentation (aerobic growth followed by anaerobic phase) resulted in enhanced succinic acid production and reduced byproduct formation. The yield of succinic acid from glucose during the anaerobic phase was 0.72 g g(-1), and the productivity was 1.01 g l(-1) h(-1).     

Use of artificial electron acceptors in oxidation reactions catalyzed by acetic acid bacteria

Summary The ability of several electron acceptors to promote the Gluconobacter oxydans catalyzed oxidation of glycerol was investigated. p-Benzoquinone was the most effective electron acceptor. The reaction rate obtained with p-benzoquinone was higher than the maximal rate with the natural electron acceptor, oxygen, in all the oxidation reactions tested.     

D-alanine oxidase from Escherichia coli: participation in the oxidation of L-alanine

Cell wall-membrane preparations of Escherichia coli, prepared by the ethylenediaminetetraacetic acid-lysozyme method, contain enzymes which catalyze the oxidation of d-alanine and, to a lesser extent, l-alanine into pyruvate and ammonia without the formation of hydrogen peroxide. The kinetic parameters were (i) pH optima of 8.3 to 8.4 for l- and d-alanine and (ii) a K(m) value of 6.6 +/- 0.2 mM for d-alanine. Several coenzymes were without effect when added to the reaction mixture. The participation of d-alanine oxidase in the oxidation of l-alanine was demonstrated. The evidence is based on (i) results of cellular fractionation; (ii) labeling experiments; (iii) inhibition studies with aminooxyacetate and cycloserine; (iv) denaturation experiments; and (v) demonstration of the presence of an active racemase.     

Studies on the mechanism of the malate dehydrogenase reaction.

The stereospecificity of the chicken heart mitochondrial malate dehydrogenase as well as the ability of this enzyme to form various abortive complexes has been further investigated. The enzyme was found to be specific for the A-hydrogen of NADH. Complex formation of the enzyme with oxalacetate and oxidized coenzymes is pH-dependent and is promoted at alkaline pH values. The enol form of oxalacetate appears to be the species that participates in the formation of the complexes. The binding of L-malate, D-malate, or hydroxymalonate to the enzyme. NADH complex is also pH-dependent, and involves a group on the enzyme with a pK of 7.5. The binding of L-malate is promoted at alkaline pH values, whereas the binding of D-malate and hydroxymalonate is favored at acidic pH values. These results indicate that L-malate and enol-oxalacetate preferentially or exclusively bind to the nonprotonated form of the enzyme, whereas keto-oxalactate, hydroxymalonate, and D-malate only bind to the protonated form of the enzyme. Based on this conclusion, a detailed chemical mechanism for the malate dehydrogenase reaction has been postulated and a schematic illustration of the transition state of the enzyme is presented.