New Insights into the Alternative d-Glucarate Degradation Pathway

Abstract

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 ϳ2450 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.

l-Hydroxyproline andd-Proline Catabolism in Sinorhizobium meliloti

“…1B. Together with other reports (12,25,59,(62)(63)(64), this report defines the genes that encode specific enzyme activities required for trans-4-hydroxy-L-proline catabolism. These studies should allow the identification of hydroxyproline catabolic genes in other organisms where many of these genes are annotated as genes of unknown function.…”

“…The ␣-KGSADH enzyme used in these reactions was either purified HypH from S. meliloti or purified ␣-KGSADH protein from Acinetobacter baylyi (25). Similar deaminase activities were detected using either enzyme.…”

“…␣-KGSA dehydrogenase enzymes involved in the catabolism of L-arabinose, D-glucarate, D-galactarate, and hydroxy-L-proline have also been characterized (25,52,53). To investigate whether the reducedgrowth phenotype of the hypH mutant on trans-4-L-Hyp may be due to the presence of other ␣-KGSADH-like enzymes, we searched the S. meliloti genome for other annotated dehydrogenases and identified the most similar as AraE, which functions as an ␣-KGSADH in the catabolism of L-arabinose (18).…”

Acinetobacter baylyi ADP1: Transforming the choice of model organism

“…Thus, NadM, which is unconditionally essential in A. baylyi, is proposed as an alternative drug target for the multidrug-resistant pathogen A. baumannii (48,49). Similar approaches were used to predict and validate a pathway to consume D-glucarate, including its regulatory mechanisms (50).…”

Purification, crystallization and preliminary X-ray diffraction analysis of a novel keto-deoxy-D-galactarate (KDG) dehydratase fromAgrobacterium tumefaciens

“…Galactarolactone cycloisomerase (EC 5.5.1.-) then catalyses ring opening and the formation of 3-deoxy-2-keto-l-threo-hexarate (Andberg et al, 2012). The 3-deoxy-2-keto-l-threo-hexarate is converted by a decarboxylating hydrolyase (EC 4.2.1.41) into -ketoglutaric semialdehyde (Jeffcoat et al, 1969;Aghaie et al, 2008). In the final step -ketoglutaric semialdehyde is oxidized by a dehydrogenase to -ketoglutarate, which is a metabolite of the TCA cycle (Watanabe et al, 2007).…”