Unusual C3 and C4 metabolism in the chemoautotroph Alcaligenes eutrophus

Abstract

Phosphoenolpyruvate (PEP) carboxykinase was identified to be the only C3-carboxylating enzyme in Alcaligenes eutrophus. The enzyme requires GDP or inosine diphosphate (GTP or inosine triphosphate) for activity. Pyruvate- and other PEP-dependent CO2-fixing enzyme activities were not detected, regardless of whether the cells were grown autotrophically or heterotrophically. It is suggested that two pathways are present in the organism for the formation of PEP from C4 dicarboxylic acids. Besides decarboxylation of oxaloacetate by PEP carboxykinase, the consecutive action of NADP+-malic enzyme and PEP synthetase can also accomplish this synthesis. An oxaloacetate decarboxylase activity observed in the cell extracts may also contribute to the latter route. The properties of a mutant deficient in PEP synthetase supported the biochemical data. This mutant was unable to grow on pyruvate or lactate and grew slower than the wild type on direct or indirect metabolites of the tricarboxylic acid cycle such as succinate, glutamate, or acetate. Growth on fructose and autotrophic growth were not affected by the enzyme defect. The findings suggest that, depending on the growth substrate utilized, PEP carboxykinase can serve a dual physiological function in A. eutrophus, an anaplerotic function in oxaloacetate synthesis from PEP, or a gluconeogenic function in PEP synthesis from oxaloacetate.

Unravelling the C 3 /C 4 carbon metabolism in Ralstonia eutropha H16

“…1). Enzyme activity assay using crude extracts of R. eutropha H16 revealed an NADP + -dependent oxidative decarboxylation of l-malate into pyruvate, whereas no NAD + -dependent reaction could be detected in this study or in the study of Schobert and Bowien (1984) ( Table 6). The heterologous expression of maeA and the subsequent determination of the enzyme activity in crude extracts of recombinant E. coli also did not reveal enzyme activity, independent of the supplied cofactor.…”

“…MaeB activity is obviously not capable of converting sufficient amounts of malate into pyruvate for gluconeogenesis. This again demonstrates the dual function and importance of Pck for replenishment of the TCC as well as for gluconeogenesis (Schobert and Bowien 1984;Brämer and Steinbüchel 2002). In addition to their gluconeogenetic and anaplerotic role, enzymes of the C3 ⁄ C4 metabolism were also shown to fulfil other important functions in bacteria metabolism.…”

The PEP—pyruvate—oxaloacetate node as the switch point for carbon flux distribution in bacteria: We dedicate this paper to Rudolf K. Thauer, Director of the Max-Planck-Institute for Terrestrial Microbiology in Marburg, Germany, on the occasion of his 65th birthday

“…The plant, fungal and almost all bacterial enzymes, in contrast, are mono-or oligomeric and use ATP as the phosphate donor (EC 4.1.1.49) [81]. Exceptions from this rule are the PEP carboxykinases of Ruminococcus flavefaciens [82], Ralstonia eutropha [83], A. globiformis [1], C. glutamicum [84] and of the hyperthermophilic archaeon Thermococcus kodakaraensis [85], which have been shown to be GTP-dependent. However, as deduced from annotated (genome) sequences, further bacteria and archaea probably also possess GTP-specific PEP carboxykinases (summarized in [85]) and thus, it might turn out that in fact many if not most bacteria possess a GTP-dependent PEP carboxykinase.…”

Metabolic Pathway for Biosynthesis of Poly (3‐Hydroxybutyrate‐co‐4‐Hydroxybutyrate) from 4‐Hydroxybutyrate by Alcaligenes eutrophus

“…Therefore, hybrid plasmid pVDZ'2: :EE10 was transferred to representative mutants, which were not able to grow with 4-hydroxybutyrate or with 4-hydroxyvalerate. Recombinant Schobert and Bowien (1984). 3HB and 4HB, strains were grown on mineral salts medium containing 0.25% (mass/vol.)…”