Dataset Information

Determination of the structure of the catabolic N-succinylornithine transaminase (AstC) from Escherichia coli.

ABSTRACT: Escherichia coli possesses two acyl ornithine aminotransferases, one catabolic (AstC) and the other anabolic (ArgD), that participate in L-arginine metabolism. Although only 58% identical, the enzymes have been shown to be functionally interchangeable. Here we have purified AstC and have obtained X-ray crystal structures of apo and holo-AstC and of the enzyme complexed with its physiological substrate, succinylornithine. We compare the structures obtained in this study with those of ArgD from Salmonella typhimurium obtained elsewhere, finding several notable differences. Docking studies were used to explore the docking modes of several substrates (ornithine, succinylornithine and acetylornithine) and the co-substrate glutamate/?-ketogluterate. The docking studies support our observations that AstC has a strong preference for acylated ornithine species over ornithine itself, and suggest that the increase in specificity associated with acylation is caused by steric and desolvation effects rather than specific interactions between the substrate and enzyme.

SUBMITTER: Newman J

PROVIDER: S-EPMC3590144 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Determination of the structure of the catabolic N-succinylornithine transaminase (AstC) from Escherichia coli.

Newman Janet J Seabrook Shane S Surjadi Regina R Williams Charlotte C CC Lucent Del D Wilding Matthew M Scott Colin C Peat Thomas S TS

PloS one 20130306 3

Escherichia coli possesses two acyl ornithine aminotransferases, one catabolic (AstC) and the other anabolic (ArgD), that participate in L-arginine metabolism. Although only 58% identical, the enzymes have been shown to be functionally interchangeable. Here we have purified AstC and have obtained X-ray crystal structures of apo and holo-AstC and of the enzyme complexed with its physiological substrate, succinylornithine. We compare the structures obtained in this study with those of ArgD from Sa ...[more]

PMID: 23484010

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Project description:BackgroundMost bacteria can use various compounds as carbon sources. These carbon sources can be either co-metabolized or sequentially metabolized, where the latter phenomenon typically occurs as catabolite repression. From the practical application point of view of utilizing lignocellulose for the production of biofuels etc., it is strongly desirable to ferment all sugars obtained by hydrolysis from lignocellulosic materials, where simultaneous consumption of sugars would benefit the formation of bioproducts. However, most organisms consume glucose prior to consumption of other carbon sources, and exhibit diauxic growth. It has been shown by fermentation experiments that simultaneous consumption of sugars can be attained by ptsG, mgsA mutants etc., but its mechanism has not been well understood. It is strongly desirable to understand the mechanism of metabolic regulation for catabolite regulation to improve the performance of fermentation.ResultsIn order to make clear the catabolic regulation mechanism, several continuous cultures were conducted at different dilution rates of 0.2, 0.4, 0.6 and 0.7 h?¹ using wild type Escherichia coli. The result indicates that the transcript levels of global regulators such as crp, cra, mlc and rpoS decreased, while those of fadR, iclR, soxR/S increased as the dilution rate increased. These affected the metabolic pathway genes, which in turn affected fermentation result where the specific glucose uptake rate, the specific acetate formation rate, and the specific CO? evolution rate (CER) were increased as the dilution rate was increased. This was confirmed by the ¹³C-flux analysis. In order to make clear the catabolite regulation, the effect of crp gene knockout (?crp) and crp enhancement (crp?) as well as mlc, mgsA, pgi and ptsG gene knockout on the metabolism was then investigated by the continuous culture at the dilution rate of 0.2 h?¹ and by some batch cultures. In the case of ?crp (and also ?mlc) mutant, TCA cycle and glyoxylate were repressed, which caused acetate accumulation. In the case of crp? mutant, glycolysis, TCA cycle, and gluconeogenesis were activated, and simultaneous consumption of multiple carbon sources can be attained, but the glucose consumption rate became less due to repression of ptsG and ptsH by the activation of Mlc. Simultaneous consumption of multiple carbon sources could be attained by mgsA, pgi, and ptsG mutants due to increase in crp as well as cyaA, while glucose consumption rate became lower.ConclusionsThe transcriptional catabolite regulation mechanism was made clear for the wild type E. coli, and its crp, mlc, ptsG, pgi, and mgsA gene knockout mutants. The results indicate that catabolite repression can be relaxed and crp as well as cyaA can be increased by crp?, mgsA, pgi, and ptsG mutants, and thus simultaneous consumption of multiple carbon sources including glucose can be made, whereas the glucose uptake rate became lower as compared to wild type due to inactivation of ptsG in all the mutants considered.

Dataset Information

Determination of the structure of the catabolic N-succinylornithine transaminase (AstC) from Escherichia coli.

Publications

Determination of the structure of the catabolic N-succinylornithine transaminase (AstC) from Escherichia coli.

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