Conserved pyridoxal protein that regulates Ile and Val metabolism

Abstract

Escherichia coli YggS is a member of the highly conserved uncharacterized protein family that binds pyridoxal 5'-phosphate (PLP). To assist with the functional assignment of the YggS family, in vivo and in vitro analyses were performed using a yggS-deficient E. coli strain (ΔyggS) and a purified form of YggS, respectively. In the stationary phase, the ΔyggS strain exhibited a completely different intracellular pool of amino acids and produced a significant amount of l-Val in the culture medium. The log-phase ΔyggS strain accumulated 2-ketobutyrate, its aminated compound 2-aminobutyrate, and, to a lesser extent, l-Val. It also exhibited a 1.3- to 2.6-fold increase in the levels of Ile and Val metabolic enzymes. The fact that similar phenotypes were induced in wild-type E. coli by the exogenous addition of 2-ketobutyrate and 2-aminobutyrate indicates that the 2 compounds contribute to the ΔyggS phenotypes. We showed that the initial cause of the keto acid imbalance was the reduced availability of coenzyme A (CoA); supplementation with pantothenate, which is a CoA precursor, fully reversed phenotypes conferred by the yggS mutation. The plasmid-borne expression of YggS and orthologs from Bacillus subtilis, Saccharomyces cerevisiae, and humans fully rescued the ΔyggS phenotypes. Expression of a mutant YggS lacking PLP-binding ability, however, did not reverse the ΔyggS phenotypes. These results demonstrate for the first time that YggS controls Ile and Val metabolism by modulating 2-ketobutyrate and CoA availability. Its function depends on PLP, and it is highly conserved in a wide range species, from bacteria to humans.

Fig 1

Structure of YggS and comparison to bacterial AR. A front view (A) and side view (B) of the YggS structure are represented. The PDB code used for YggS is 1W8G. (C) The superposition of the YggS structure onto that of the monomeric structure of G. stearothermophilus AR (PDB entry 1SFT). (D and E) Stereo view of the putative active site of YggS (D) and the active site of AR (E).

Fig 2

Purity of the recombinant YggS and the UV-visible spectra in the presence or absence of d- and l-Cys. (A) The purified protein was separated on a 12% SDS-PAGE gel. MW, molecular weight (in thousands). (B) Absorption spectra of the purified YggS in the absence (line 1) or presence of d-Cys (5 mM; line 2) and l-Cys (5 mM; line 3). These spectra were recorded in PBS buffer at a protein concentration of 31 μM.

Fig 3

Growth of WT and ΔyggS strains in the M9-glucose medium. Wild-type (•) and ΔyggS (○) strains were cultivated in the M9-glucose medium at 30°C. The OD₆₀₀ was determined 4, 7, 10, 13, and 16 h after inoculation.

Fig 4

Inhibitory effect of ΔyggS mutant strain culture supernatant. Wild-type E. coli MG1655 (shown as the WT), E. coli MG1655 ΔyggS mutant (ΔyggS), E. coli BL21 (BL21), and E. coli XL1-Blue (XL1 blue) were grown in the M9-glucose medium containing 24-h-old WT or ΔyggS culture supernatant (sup.). The 24-h-old culture supernatants were filter sterilized and added to a freshly prepared M9-glucose medium at a final concentration of 25%. l-Ile and l-Leu were added at a final concentration of 0.1 mM. After 24 h of cultivation, the OD₆₀₀ was determined.

Fig 5

(A) Elution profile of extracellular amino acid at the stationary phase. Wild-type and ΔyggS mutant strains were grown in M9-glucose medium at 30°C. The stationary-phase culture supernatants were collected, deproteinized, and subjected to amino acid analysis. The HPLC chromatograms generated by the amino acid analysis are shown (wild-type, solid line; ΔyggS mutant, broken line). The inset graph shows an expansion of the retention time from 25 to 40 min. The extracellular l-Val (V) concentration was 0.7 and 110 μM in the WT and ΔyggS mutant culture supernatant, respectively. The ΔyggS mutant produced 2-aminobutyrate (*), and only the WT accumulated extracellular Nle (#). (B) Intracellular amino acid composition of stationary-phase cells. Both WT (closed bar) and ΔyggS (open bar) strains were grown in M9-glucose medium at 30°C. After 24 h of cultivation, the concentrations of intracellular amino acids were determined as described in Materials and Methods.

Fig 6

Intracellular amino acid composition of the log-phase WT (closed bar) and ΔyggS (open bar) strains. Both WT and ΔyggS strains were grown in M9-glucose medium at 30°C. The log-phase cells (OD₆₀₀, 0.5) were collected and used for the amino acid analysis. 2-AB, 2-aminobutyrate.

Fig 7

Effect of 2-ketobutyrate, 2-aminobutyrate, and pantothenate on l-Val productivity. The HPLC chromatograms generated by amino acid analysis of the stationary-phase culture supernatant of WT (left) and ΔyggS mutant (right) strains cultivated alone (A and E) or in the presence of 1 mM 2-ketobutyrate (B and F), 1 mM 2-aminobutyrate (C and G), and 1 mM pantothenate (D and H). The cells were grown in the M9-glucose medium at 30°C. Symbols: *, 2-aminobutyrate; V, valine; I, isoleucine; #, norleucine.

Fig 8

Schematic view of the l-Ile and l-Val metabolic pathways in E. coli. Metabolites and enzymes (underlined) in Ile and Val metabolism are shown. The symbols (Δ or ▼) indicate an increase (Δ) or decrease (▼) in the level of the compound or the enzyme in log phase. 2-AB, 2-aminobutyrate; 2-KB, 2-ketobutyrate; Pyr, pyruvate; Pan, pantothenate; α-AL, α-acetolactate; α-AHB, α-aceto-α-hydroxybutyrate; α-KMV, α-keto-β-methylvalerate; α-KIV, α-ketoisovalerate; α-KG, α-ketoglutarate.

Fig 9

Total CoA levels in WT and ΔyggS mutant strains. Strains were grown using M9-glucose medium at 30°C. Total CoA levels were quantified according the protocol described in Materials and Methods. 2-Ketobutyrate was added to the medium at a final concentration of 0.1 mM.

Fig 10

Effect on l-Val productivity of the yggS mutation and the introduction of YggS orthologs. The WT harboring a control vector (pU0 and pGEX-4T-1) and the ΔyggS strain harboring pU0, pGEX-4T-1, pUS, pUSm, pGEX-yggS, pGEX-YBL036C, pGEX-prosc, or pGEX-ylmE were cultivated in M9-glucose medium at 30°C. pUSm expresses a YggS mutant (K36A) lacking PLP binding ability. The l-Val concentration of the stationary-phase culture supernatant was estimated with leucine dehydrogenase as described in Materials and Methods.