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.In
Escherichia coli, the Entner-Doudoroff (ED) pathway serves as a metabolic “funnel” receiving intermediates formed by catabolism of several sugar acids (
17). Hexuronic acids undergo rearrangement in the inducible Ashwell pathways (
1) to form 2-keto-3-deoxygluconate, which is then phosphorylated to produce 2-keto-3-deoxy-6-phosphogluconate (KDPG). KDPG is cleaved by KDPG aldolase, encoded by
eda, providing for entry of carbon into glycolysis. The other enzyme of the ED pathway is 6-phosphogluconate dehydratase, encoded by
edd, which is induced only for catabolism of gluconate and also forms KDPG, the key intermediate of the ED pathway (
7). Long considered to be of more significance than is readily obvious (
9), the finding that
eda and
edd eda double mutants are unable to colonize the mouse large intestine underscores the possible ecological importance of ED metabolism (
32). The implication from these colonization studies is that colonic mucus, which contains several sugar acids, may serve as an important source of nutrients for
E. coli in the gut.Also participating in gluconate catabolism are several gluconate transporters and two gluconate kinases which appear, based upon their regulation, to comprise two distinct systems (
2,
13). The GntI (main) system consists of
gntT,
gntU, and
gntK, which code for high- and low-affinity gluconate transporters and a thermoresistant gluconate kinase, respectively (
23–
25,
33). Expression of the GntR regulon, that is, GntI together with the
edd-eda operon, is negatively controlled by the
gntR gene product. The GntII (subsidiary) system is comprised of a thermosensitive gluconate kinase and a gluconate transporter which function for gluconate catabolism in the absence of the GntI system (
2,
11,
13,
22). It appears that the subsidiary gluconate transporter, which has an apparent
Km for gluconate of 60 μM (
23), is encoded by a gene (
idnT) which is adjacent to the gene encoding the thermosensitive gluconokinase (
idnK) at 96.8 min.The DNA sequence of the GntII system genes, located at 4492 kb on the genome, was revealed by the
E. coli Genome Project (
5,
6). If the GntII system had evolved as a subsidiary pathway for gluconate catabolism, one would expect it to contain only a gluconate transporter and gluconate kinase. However, in addition to the divergent
idnK and
idnT genes, this region also encodes two “dehydrogenase-like” enzymes. The similarity of
idnO to
gno of
Gluconobacter oxydans, which encodes
d-gluconate:NADP 5-oxidoreductase (GNO) (
15), led to the testing of ketogluconates as enzyme substrates for the two newly identified dehydrogenases. A process of deductive reasoning and biochemical experiments led to the conclusion that the GntII system in fact comprises a novel metabolic pathway for catabolism of
l-idonic acid, in which gluconate is a key intermediate. Accordingly, the genes involved in
l-idonate metabolism have been given the designation
idn (see Table for gene nomenclature).
TABLE 1
Gene and enzyme nomenclature
a Gene designation
| Gene product | % Identity of proteinb |
---|
Previous | New (acces- sion no.) |
---|
gntV | idnK ({"type":"entrez-protein","attrs":{"text":"P39208","term_id":"729610","term_text":"P39208"}}P39208) | d-Gluconate kinase | 45 (GntKc) |
yjgV | idnD ({"type":"entrez-protein","attrs":{"text":"P39346","term_id":"732054","term_text":"P39346"}}P39346) | l-Idonate 5-dehydrogenase | 30.6 (sheep DHSOd) |
yjgU | idnO ({"type":"entrez-protein","attrs":{"text":"P39345","term_id":"732053","term_text":"P39345"}}P39345) | 5-Keto-d-gluconate 5-reductase | 56 (GNOe) |
gntW | idnT ({"type":"entrez-protein","attrs":{"text":"P39344","term_id":"732052","term_text":"P39344"}}P39344) | l-Idonate transporter | 61 (GntTf) |
yjgS | idnR ({"type":"entrez-protein","attrs":{"text":"P39343","term_id":"732051","term_text":"P39343"}}P39343) | l-Idonate regulator | 46 (GntRg) |
Open in a separate windowaAll accession numbers are Swiss-Prot database accession numbers.
bPercent identity of the amino acid sequence of the Idn protein to that of the protein shown in parentheses.
cE. coli gluconate kinase encoded by
gntK (
{"type":"entrez-protein","attrs":{"text":"P46859","term_id":"2507037","term_text":"P46859"}}P46859).
dSheep sorbitol dehydrogenase encoded by
sorD (
{"type":"entrez-protein","attrs":{"text":"P07846","term_id":"118625","term_text":"P07846"}}P07846).
eG. oxydans gluconate:NADP 5-oxidoreductase encoded by
gno (
{"type":"entrez-protein","attrs":{"text":"P50199","term_id":"1708001","term_text":"P50199"}}P50199).
fE. coli gluconate transporter encoded by
gntT (
{"type":"entrez-protein","attrs":{"text":"P39835","term_id":"54041238","term_text":"P39835"}}P39835).
gE. coli gluconate regulator encoded by
gntR (
{"type":"entrez-protein","attrs":{"text":"P46860","term_id":"2506561","term_text":"P46860"}}P46860). (Part of this work has been presented previously [
3].)
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