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Notes: The labelling patterns of metabolites from experiments with stable isotope-labelled precursors can be determined by NMR spectroscopy. Complex isotopomer mixtures are found when general metabolites such as glucose are used as stable isotope-labelled precursors which are diverted to all branches of intermediary metabolism. The complex results can be interpreted by a pattern recognition approach based on comparison between the labelling patterns of secondary metabolites and primary metabolites such as amino acids and ribonucleosides. The isotope labelling patterns of intermediates in central metabolic pools such as carbohydrate phosphates, dicarboxylic acids, and acetyl CoA can be obtained by biosynthetic retroanalysis. Biosynthetic pathways as well as metabolite flux patterns can be determined from these data. The method is illustrated using the classical mevalonate pathway and the more recently discovered deoxyxylulose pathway of terpenoid biosynthesis as examples. Applications of the retrobiosynthetic method of the biosynthesis of molybdopterin and of riboflavin are also discussed. Stable isotope experiments monitored by NMR spectroscopy have also been shown to be a powerful tool for the elucidation of metabolic flux in microorganisms with unusual lifestyles and in fermentation processes.

Notes: Aromatic compounds are important growth substrates for microorganisms. They form a large group of diverse compounds including lignin monomers, amino acids, quinones, and flavonoids. Aerobic aromatic metabolism is characterized by the extensive use of molecular oxygen which is essential for the hydroxylation and cleavage of aromatic ring structures. The anaerobic metabolism of low molecular mass soluble aromatic compounds requires, of necessity, a quite different strategy. In most known cases, aromaticity is broken by reduction and the ring is subsequently opened hydrolytically. A small number of different central aromatic intermediates can be reduced, the most common of which is benzoyl-CoA, a compound that is formed as a central intermediate in the degradation of a large number of aromatic growth substrates. This review concentrates on the anaerobic aromatic metabolism via the benzoyl-CoA pathway. The peripheral pathways that transform growth substrates to benzoyl-CoA include various types of novel reactions, for example carboxylation of phenolic compounds, reductive elimination of ring substituents like hydroxyl or amino groups, oxidation of methyl substituents, O-demethylation reactions and shortening of aliphatic side chains. The central benzoyl-CoA pathway differs in several aspects in the denitrifying, phototrophic and fermenting bacteria studied. In denitrifying and phototrophic bacteria it starts with the two-electron reduction of benzoyl-CoA to a cyclic dienoyl-CoA driven by the hydrolysis of two molecules of ATP to ADP+Pi. This ring reduction is catalyzed by benzoyl-CoA reductase and requires a low-potential ferredoxin as an electron donor. In Rhodopseudomonas palustris the cyclic diene is further reduced to cyclohex-1-ene-1-carboxyl-CoA. In the denitrifying species Thauera aromatica, the cyclic diene is hydrated to give 6-hydroxycyclohex-1-ene-1-carboxyl-CoA. Subsequent β-oxidation results in the formation of a cyclic β-oxo compound, followed by hydrolytic carbon ring opening yielding 3-hydroxypimelyl-CoA in the case of T. aromatica and pimelyl-CoA in the case of R. palustris. These intermediates are further β-oxidized via glutaryl-CoA; final products are 3 acetyl-CoA and 1 CO2. In fermenting bacteria benzoyl-CoA may possibly be reduced to the level of cyclohex-1-ene-1-carboxyl-CoA in an ATP-independent reaction. The genes coding for the enzymes of the central benzoyl-CoA pathway have been cloned and sequenced from R. palustris, T. aromatica, and Azoarcus evansii. Sequence analyses of the genes support the concept that phototrophic and denitrifying bacteria use two slightly different pathways to metabolize benzoyl-CoA. The gene sequences have in some cases been very helpful for the identification of possible catalytic mechanisms that were not obvious from initial characterizations of purified enzymes.

Notes: The stable carbon isotopic compositions of the inorganic carbon source, bulk cell material, and isoprenoid lipids of the hyperthermophilic crenarchaeon Metallosphaera sedula, which uses a 3-hydroxypropionate-like pathway for autotrophic carbon fixation, have been measured. Bulk cell material was approximately 3‰ enriched in 13C relative to the dissolved inorganic carbon, and 2‰ depleted in 13C relative to isoprenoid membrane lipids. The isotope data suggested that M. sedula uses mainly bicarbonate rather than CO2 as inorganic carbon source, which is in accordance with a 3-hydroxypropionate-like carbon fixation pathway. To the best of our knowledge this is the first report of 13C fractionation effects of such a hyperthermophilic crenarchaeon.

Notes: A new principle of aerobic aromatic metabolism has been postulated, which is in contrast to the known pathways. In various bacteria the aromatic substrate benzoate is first converted to its coenzyme A (CoA) thioester, benzoyl-CoA, which is subsequently attacked by an oxygenase, followed by a non-oxygenolytic fission of the ring. We provide evidence for this hypothesis and show that benzoyl-CoA conversion in the bacterium Azoarcus evansii requires NADPH, O2 and two protein components, BoxA and BoxB. BoxA is a homodimeric 46 kDa iron-sulphur-flavoprotein, which acts as reductase. In the absence of BoxB, BoxA catalyses the benzoyl-CoA stimulated artificial transfer of electrons from NADPH to O2 via free FADH2 to produce H2O2. Physiologically, BoxA uses NADPH to reduce BoxB, a monomeric 55 kDa iron-protein that acts as benzoyl-CoA oxygenase. The product of benzoyl-CoA oxidation was identified by NMR spectroscopy as its dihydrodiol derivative, 2,3-dihydro-2,3-dihydroxybenzoyl-CoA. This suggests that BoxBA act as a benzoyl-CoA dioxygenase/reductase. Unexpectedly, benzoyl-CoA transformation by BoxBA was greatly stimulated when another enoyl-CoA hydratase/isomerase-like protein, BoxC, was added that catalysed the further transformation of the dihydrodiol product formed from benzoyl-CoA. The benzoyl-CoA oxygenase system has very low similarity to known (di)oxygenase systems and is the first member of a new enzyme family.

Notes: A novel aerobic benzoate pathway has recently been discovered in various bacteria in which benzoate is first converted to benzoyl-CoA. The further downstream steps are associated with the gene products of the benzoate oxidation gene cluster (box) on the Azoarcus evansii chromosome. Benzoyl-CoA is oxidized to 2,3-dihydro-2,3-dihydroxybenzoyl-CoA (benzoyl-CoA dihydrodiol) by benzoyl-CoA oxygenase/reductase BoxBA in the presence of molecular oxygen. This study identified the next, ring cleaving step catalysed by BoxC. The boxC gene was expressed in a recombinant Escherichia coli strain as a fusion protein with maltose binding protein (BoxCmal) and the wild type as well as the recombinant proteins were purified and studied. BoxC catalyses the reaction 2,3-dihydro-2,3-dihydroxybenzoyl-CoA + H2O → 3,4-dehydroadipyl-CoA semialdehyde + HCOOH. This is supported by the following results. Assays containing [ring-13C6]benzoyl-CoA, benzoyl-CoA oxygenase/reductase, BoxCmal protein, NADPH and semicarbazide were analysed directly by NMR spectroscopy and mass spectrometry. The products were identified as the semicarbazone of [2,3,4,5,6-13C5]3,4-dehydroadipyl-CoA semialdehyde; the missing one-carbon unit being formate. The same reaction mixture without semicarbazide yielded a mixture of the hydrate of [2,3,4,5,6-13C5]3,4-dehydroadipyl-CoA semialdehyde and [2,3,4,5,6-13C5]4,5-dehydroadipyl-CoA semialdehyde. BoxC, a 122 kDa homodimeric enzyme (61 kDa subunits), is termed benzoyl-CoA-dihydrodiol lyase. It contains domains characteristic for enoyl-CoA hydratases/isomerases, besides a large central domain with no significant similarity to sequences in the database. The purified protein did not require divalent metals, molecular oxygen or any cosubstrates or coenzymes for activity. The complex reaction is part of a widely distributed new principle of aerobic aromatic metabolism in which all intermediates are coenzyme A thioesters and the actual ring-cleavage reaction does not require molecular oxygen.

Notes: Abstract Permeabilized cells of Desulfovibrio baarsii catalyzed an isotopic exchange between 14CO and the carboxyl group of acetyl-CoA and an isotopic exchange between 14CO2 and the carboxyl group of acetyl-CoA. Cyanide, which inactivated the carbon monoxide dehydrogenase activity present in the cells, inhibited the 14CO2/acetyl-CoA rather than the 14CO/acetyl-CoA exchange reaction.

Notes: Abstract An increasing number of strict anaerobic bacteria are being found which use an alternative pathway to the ubiquitous Calvin cycle for CO2 fixation into cell compounds and the ubiquitous Krebs cycle for acetyl CoA oxidation to CO2. The principles of this non-cyclic pathway, the acetyl CoA pathway, have long been studied in acetogenic bacteria. These bacteria can catalyze the exergonic reduction of 2 CO2 with 8 reducing equivalents to acetate. In this pathway, CO2 reduction is part of a catabolic redox process which functions to accept reducing equivalents from a variety of dehydrogenated substrates. This process yields net ATP generated by electron transport phosphorylation. Acetyl CoA is an intermediate, formed from one CO2 via a tetrahydropteridine-bound 1-carbon unit (methyl group of acetate), and from another CO2 via a bound carbon monoxide (carboxyl group of acetate). The most characteristic and complex enzyme involved in acetyl CoA synthesis is carbon monoxide dehydrogenase (‘acetyl CoA synthase’). The enzymes of this acetyl CoA pathway not only participate in (1) acetate synthesis in energy metabolism of acetogenic bacteria, but also mediate (2) acetyl CoA oxidation in sulfate-reducing bacteria and possibly other anaerobes; (3) acetate disproportionation to CO2 and CH4 in the energy metabolism of many methanogenic bacteria; (4) autotrophic CO2 fixation in autotrophic acetogenic, methanogenic, and most autotrophic sulfate-reducing bacteria; (5) assimilation and/or dissimilation of 1-carbon compounds in many anaerobes; (6) CO oxidation to CO2 in anaerobes. A specialized group of anaerobes performs acetate synthesis from CO2 or from C1 units via a different pathway, the glycine synthase/glycine reductase pathway. Glycine is an intermediate which is formed from 2 C1 compounds, and is then reduced to acetate. The principal features of the two pathways and some open questions are discussed in this review. Emphasis is placed upon the acetyl CoA pathway in acetogenic bacteria, but important advances in the study of other strict anaerobes are also considered.