Sequence analysis of the GntII (subsidiary) system for gluconate metabolism reveals a novel pathway for L-idonic acid catabolism in Escherichia coli

Abstract

The presence of two systems in Escherichia coli for gluconate transport and phosphorylation is puzzling. The main system, GntI, is well characterized, while the subsidiary system, GntII, is poorly understood. Genomic sequence analysis of the region known to contain genes of the GntII system led to a hypothesis which was tested biochemically and confirmed: the GntII system encodes a pathway for catabolism of L-idonic acid in which D-gluconate is an intermediate. The genes have been named accordingly: the idnK gene, encoding a thermosensitive gluconate kinase, is monocistronic and transcribed divergently from the idnD-idnO-idnT-idnR operon, which encodes L-idonate 5-dehydrogenase, 5-keto-D-gluconate 5-reductase, an L-idonate transporter, and an L-idonate regulatory protein, respectively. The metabolic sequence is as follows: IdnT allows uptake of L-idonate; IdnD catalyzes a reversible oxidation of L-idonate to form 5-ketogluconate; IdnO catalyzes a reversible reduction of 5-ketogluconate to form D-gluconate; IdnK catalyzes an ATP-dependent phosphorylation of D-gluconate to form 6-phosphogluconate, which is metabolized further via the Entner-Doudoroff pathway; and IdnR appears to act as a positive regulator of the IdnR regulon, with L-idonate or 5-ketogluconate serving as the true inducer of the pathway. The L-idonate 5-dehydrogenase and 5-keto-D-gluconate 5-reductase reactions were characterized both chemically and biochemically by using crude cell extracts, and it was firmly established that these two enzymes allow for the redox-coupled interconversion of L-idonate and D-gluconate via the intermediate 5-ketogluconate. E. coli K-12 strains are able to utilize L-idonate as the sole carbon and energy source, and as predicted, the ability of idnD, idnK, idnR, and edd mutants to grow on L-idonate is altered.

FIG. 1

Enzymes and genes of l-idonate catabolism. Abbreviations: 6-P-Gluconate, 6-phosphogluconate; L-Ido, l-idonate; 5K-D-Gnt 5-Red, 5-keto-d-gluconate 5-reductase; L-Ido 5-DeHase, l-idonate 5-dehydrogenase; Gnt, gluconate.

FIG. 2

HPLC analysis of substrate conversion. Conversion from 5KG to d-gluconate by 5-keto-d-gluconate 5-reductase (A), from 5KG to l-idonate by l-idonate 5-dehydrogenase (B), and from 5KG to both l-idonate and d-gluconate by 5-keto-d-gluconate 5-reductase and l-idonate 5-dehydrogenase (C) are shown. Authentic l-idonate standard (D) is also shown.

FIG. 3

Partial ¹H NMR spectra of sodium d-gluconate (A), sodium l-idonate (B), and an unknown compound (C) isolated from reaction of l-idonate 5-dehydrogenase with 5KG and NADH, indicating that the unknown compound is l-idonate. Spectra were recorded at 500 MHz in D₂O.

FIG. 4

Growth of E. coli on l-idonate, d-gluconate, and d-glucose. E. coli W1485 (open circles), E. coli BM129 (edd mutant; open squares), and E. coli NP250 (idnR mutant; closed circles) grown on M63 minimal medium containing l-idonate, d-gluconate, and d-glucose are shown.