The alternative d-glucarate degradation pathway proposed in Pseudomonas. Shown are d-glucarate dehydratase (1), d-5-keto-4-deoxyglucarate dehydratase (2), andα-ketoglutarate semialdehyde dehydrogenase (3).

Comparison of the steady-state kinetic behavior of α-KGSA and propanal oxidation by α-KGSA dehydrogenase of A. baylyi ADP1, in the presence of 100μm NADP⁺ in Activity Buffer. A, plot of v versus [S] forα-KGSA oxidation, with a line drawn using the SigmaPlot software (Systat Software, Inc.) and Equation 1. B, Eadie-Hofstee representation (v versus v/[S]) of the values presented in A. α-KGSA oxidation experiments were performed with an enzyme concentration of 0.25 μg/ml. C, plot of v versus [S] for propanal oxidation, with a line drawn using the Michaelis-Menten equation. D, Eadie-Hofstee representation (v versus v/[S]) of the values presented in C. Propanal oxidation experiments were performed with an enzyme concentration of 6 μg/ml.

Transcriptional and enzymatic analysis of d-glucarate degradation in A. baylyi ADP1. A, diagram of gene clusters involved in d-glucarate/d-galactarate degradation. For each medium, genes are present if evidence of co-transcription of two consecutive genes was found. nd, the experiment was not performed. The black line at the top (P) refers to the 373-bp region located between ACIAD0127 and ACIAD0128 containing the putative operon promoter. B, results of the quantitative PCR analysis. Two independent experiments were considered except for ACIAD0126. The induction reported on the y axis is calculated as 2ⁿ (where n represents the difference in number of PCR cycles required to reach a defined amount of PCR product between RNA extracted from the wild type (WT) grown on succinate and the strain/grown under the conditions tested). Light gray, gray, and black boxes correspond to experiments performed with wild type grown on d-glucarate, ΔACIAD0132 grown on succinate, and ΔACIAD0132 grown on d-glucarate, respectively. C, α-KGSA dehydrogenase activity measured in cell extracts. Values correspond to the average of three replicates. Light gray and gray boxes represent experiments triggered with 1.5 mm d-glucarate and 200 μm α-KGSA, respectively. Experiments done with d-glucarate were conducted in Activity Buffer containing 5 mm MgCl₂ and 100 μm NADP⁺. Activities are expressed in pmol of α-ketoglutarate/min/mg of protein.