New Insights into the Alternative D-Glucarate Degradation Pathway

Aghaie, Asadollah; Lechaplais, Christophe; Sirven, Peggy; Tricot, Sabine; Besnard-Gonnet, Marielle; Muselet, Delphine; de Berardinis, Véronique; Kreimeyer, Annett; Gyapay, Gabor; Salanoubat, Marcel; Perret, Alain

New Insights into the Alternative D-Glucarate Degradation Pathway

2008

Although the D-glucarate degradation pathway is well characterized in Escherichia coli, genetic and biochemical information concerning the alternative pathway proposed in Pseudomonas species and Bacillus subtilis remains incomplete. Acinetobacter baylyi ADP1 is a Gram-negative soil bacterium possessing the alternative pathway and able to grow using D-glucarate as the only carbon source. Based on the annotation of its sequenced genome (1), we have constructed a complete collection of singlegene deletion mutants (2). High throughput profiling for growth on a minimal medium containing D-glucarate as the only carbon source for ~2450 mutants led to the identification of the genes involved in D-glucarate degradation. Protein purification after recombinant production in E. coli allowed us to reconstitute the enzymatic pathway in vitro. We describe here the kinetic characterization of D-glucarate dehydratase, D-5-keto-4-deoxyglucarate dehydratase, and of cooperative α-ketoglutarate semialdehyde dehydrogenase. Transcription and expression analyses of the genes involved in D-glucarate metabolism within a single organism made it possible to access information regarding the regulation of this pathway for the first time.

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