New Insights into the Alternative d-Glucarate Degradation Pathway
Résumé
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 -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.