New insights into the alternative D-glucarate degradation pathway.
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 approximately 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 alpha-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.
Aghaie
Asadollah
A
CNRS-UMR 8030, Genoscope-Commissariat à l'Energie Atomique, 2 Rue Gaston Crémieux, Evry 91057, France.
Lechaplais
Christophe
C
Sirven
Peggy
P
Tricot
Sabine
S
Besnard-Gonnet
Marielle
M
Muselet
Delphine
D
de Berardinis
Véronique
V
Kreimeyer
Annett
A
Gyapay
Gabor
G
Salanoubat
Marcel
M
Perret
Alain
A
D000150:Acinetobacter / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D016680:Genome, Bacterial / Q000502:physiology; D005977:Glutarates / Q000378:metabolism*; D006836:Hydro-Lyases / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic / Q000502:physiology
D001426:Bacterial Proteins; D005977:Glutarates; D011994:Recombinant Proteins; D006836:Hydro-Lyases; C020572:glucarate dehydratase