Reduced transaminase B (IlvE) activity caused by the lack of yjgF is dependent on the status of threonine deaminase (IlvA) in Salmonella enterica serovar Typhimurium

Abstract

The YjgF/YER057c/UK114 family is a highly conserved class of proteins that is represented in the three domains of life. Thus far, a biochemical function demonstrated for these proteins in vivo or in vitro has yet to be defined. In several organisms, strains lacking a YjgF homolog have a defect in branched-chain amino acid biosynthesis. This study probes the connection between yjgF and isoleucine biosynthesis in Salmonella enterica. In strains lacking yjgF the specific activity of transaminase B, catalyzing the last step in the synthesis of isoleucine, was reduced. In the absence of yjgF, transaminase B activity could be restored by inhibiting threonine deaminase, the first enzymatic step in isoleucine biosynthesis. Strains lacking yjgF showed an increased sensitivity to sulfometruron methyl, a potent inhibitor of acetolactate synthase. Based on work described here and structural reports in the literature, we suggest a working model in which YjgF has a role in protecting the cell from toxic effects of imbalanced ketoacid pools.

FIG. 1.

Isoleucine and valine biosynthetic pathways. The biosynthetic pathways for isoleucine and valine are schematically represented. Genes whose products catalyze the reactions are listed above their respective arrows. Although IlvGM and IlvBN are acetolactate synthase isozymes, IlvBN contributes primarily to Val biosynthesis, and IlvGM contributes to both pathways in cultures grown in glucose (1). Abbreviations: THR, l-threonine; KB, AKB; AHB, 2-aceto-2-hydroxybutyrate; DHMV, 2,3-dihyroxy-3-methylvalerate; KMV, 2-keto-3-methylvalerate; ILE, l-isoleucine; PYR, pyruvate; AL, 2-acetolactate; DHIV, 2,3-dihydroxyisovalerate; KIV, 2-ketoisovalerate; VAL, l-valine.

FIG. 2.

Distinct specific activity is inherent in purified transaminase B. Transaminase B protein was expressed and purified from yjgF mutant (DM6970) and wild-type (DM6971) strain backgrounds. (A) The proteins purified from DM6970 (1) and the wild-type strain (2) were assayed, and the specific activity is shown. (B) Aliquots containing 200 ng of the protein preparations were run in a 12.5% acrylamide gel to assess purity and potential mobility differences. A protein ladder standard was loaded in lane C. The minor band in the doublet is the uncleaved fusion protein that is the initial product of the construct.

FIG. 3.

Overexpression of ilvE does not restore transaminase activity in yjgF mutants. IlvE specific activity was determined in permeabilized cell extracts of wild-type and yjgF mutant strains carrying either empty vector pSU19 or pIlvE-G1. Strains were grown in minimal glucose medium containing 2.5 μg of chloramphenicol/ml and harvested, and transaminase B activity was assayed. Bars A to D represent strains grown in minimal medium. Bars E to H represent strains grown in minimal medium containing Ile. The strains, genotypes, and bar designations are as follows: A and E, DM6735 (yjgF/pSU19); B and F, DM6738 (wt/pSU19); C and G, DM6736 (yjgF/pIlvE-G1); and D and H, DM6739 (wt/pIlvE-G1). Data from the strains carrying a yjgF mutation are marked with hatched bars.

FIG. 4.

Working model for the role of YjgF in Ile biosynthesis. The working model for YjgF function with respect to Ile biosynthesis is represented. Specific features of the model are described in the text (see Discussion). This model makes no conclusions about the identity of compound X and considers that it could be AKB, a metabolite of AKB, or a distinct 2-ketoacid.