New Insights into the Alternative d-Glucarate Degradation Pathway

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

doi:10.1074/jbc.m800487200

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 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.

Highlights

Including Pseudomonas species, in which the enzymes of the pathway have been characterized [9, 11, 12] (Fig. 1)
KDG is converted to ␣-ketoglutarate by the sequential action of KDG dehydratase (EC 4.2.1.41) and ␣-ketoglutarate semialdehyde dehydrogenase (␣-KGSA dehydrogenase; EC 1.2.1.26)
Finding Genes Involved in D-Glucarate/D-Galactarate Degradation—The growth of the whole mutant collection was investigated on liquid MA minimal medium containing either succinate or D-glucarate as the sole carbon source

Summary

Introduction

Including Pseudomonas species, in which the enzymes of the pathway have been characterized [9, 11, 12] (Fig. 1). KDG is converted to ␣-ketoglutarate by the sequential action of KDG dehydratase (EC 4.2.1.41) and ␣-ketoglutarate semialdehyde dehydrogenase (␣-KGSA dehydrogenase; EC 1.2.1.26) The information on this pathway is scattered and incomplete, limiting the understanding of D-glucarate metabolism. We took advantage of the availability of the complete collection of single-gene deletion mutants to identify the genes involved in the utilization of D-glucarate by profiling ϳ2450 mutants for growth on a medium with D-glucarate as the sole carbon source. Based on this strategy, we describe here the enzymatic and transcriptional analysis of proteins and genes involved in D-glucarate utilization in A. baylyi ADP1

Methods

Results

Conclusion