Reduction of Feedback Inhibition in Homoserine Kinase (ThrB) of Corynebacterium glutamicum Enhances l-Threonine Biosynthesis

Abstract

l-Threonine is an important supplement in the food industry. It is currently produced through fermentation of Escherichia coli but requires additional purification steps to remove E. coli endotoxin. To avoid these steps, it is desirable to use Corynebacterium glutamicum, a microorganism generally regarded as safe. Engineering of C. glutamicum to increase production of l-threonine has mainly focused on gene regulation as well as l-threonine export or carbon flux depletion. In this study, we focus on the negative feedback inhibition produced by l-threonine on the enzyme homoserine kinase (ThrB). Although l-threonine binds to allosteric sites of aspartate kinase (LysC) and homoserine dehydrogenase (Hom), serving as a noncompetitive inhibitor, it acts as a competitive inhibitor on ThrB. This is problematic when attempting to engineer enzymes that are nonresponsive to increasing cellular concentrations of l-threonine. Using primary structure alignment as well as analysis of the Methanocaldococcus jannaschii ThrB (MjaThrB) active site in complex with l-threonine (inhibitor of ThrB) and l-homoserine (substrate of ThrB), a conserved active-site alanine residue (A20) in C. glutamicum ThrB (CglThrB) was predicted to be important for differential interactions with l-threonine and l-homoserine. Through site-directed mutagenesis, we show that one variant of C. glutamicum ThrB, CglThrB-A20G, retains wild-type enzymatic activity, with dramatically decreased feedback inhibition by l-threonine. Additionally, by solving the first Corynebacterium X-ray crystal structure of homoserine kinase, we can confirm that the changes in l-threonine affinity to the CglThrB-A20G active site derive from loss of van der Waals interactions.

Figure 1

Biosynthetic pathway of l-threonine and its regulation in C. glutamicum. l-Threonine regulates its own biosynthetic pathway by allosterically inhibiting aspartate kinase (LysC) and homoserine dehydrogenase (Hom) in a noncompetitive manner and homoserine kinase (ThrB) as a competitive inhibitor (shown in broken lines). Solid lines indicate branch pathways related to the l-threonine biosynthetic pathway.

Figure 2

(A) Alignment of C. glutamicum homoserine kinase (CglThrB) sequence with those of E. coli, M. jannaschii, and M. tuberculosis using CLUSTAL O (1.2.4). Conserved residues are shown with asterisk (*), residues with strongly similar properties are shown with a colon (:), and residues with weakly similar properties are shown with a period (.). The conserved Ala residues in motif I are highlighted in yellow. The arrows represent β-strands, whereas the ovals represent α-helices. (B) Structure of C. glutamicum ThrB (CglThrB-WT) with modeled l-homoserine and AMP-PNP based on the structure of M. jannaschii ThrB, with these compounds bound in the active site.

Figure 3

Interactions of C. glutamicum ThrB (A in cyan) or M. jannaschii ThrB (B and C in green) between binding site amino acids and substrates or ligands. (A) A phosphate anion is bound in the active site of CglThrB-WT, preventing binding of substrates or inhibitors within the amino acid binding site. (B) l-homoserine bound into the active site of M. jannaschii ThrB (PDB: 1H72). (C) l-threonine bound into the active site of M. jannaschii ThrB (PDB: 1H73).

Figure 4

Enzymatic activity of WT and four variants of CglThrB. CglThrB-A20G is as active as CglThrB-WT. The concentration of ADP (Y axis) was determined after incubating each enzyme with l-homoserine and ATP at 27 °C for 30 min. WT: CglThrB-WT, A20G: CglThrB-A20G, A20V: CglThrB-A20V, A20L: CglThrB-A20L, and A20S: CglThrB-A20S.

Figure 5

Kinetic reaction rates in the presence of 0, 10, 20, and 50 mM l-threonine of CglThrB-WT and CglThrB-A20G variant. Each datum represents the value of three determinations (n = 3). l-Threonine concentration: ●: 0 mM, ○: 10 mM, ▼: 20 mM, and ∇: 50 mM.

Figure 6

CglThrB-A20G has eliminated much of the feedback inhibition while retaining wild-type enzymatic activity. The reaction conditions were the same as those in Figure 4 except that different concentrations of l-threonine (0, 5, 10, and 20 mM) were used.

Figure 7

Hydrolyzed structure of CglThrB-WT (gold) superimposed with the full-length CglThrB-WT (cyan) containing modeled l-homoserine and AMP-PNP from 1H72.

Figure 8

Significant shift of the lower lip that interacts with both magnesium and the amino acid present in the active site. (A) M. jannaschii ThrB-WT with l-isoleucine, magnesium (purple sphere) and ATP-γS bound into its active site (PDB: 1H74). (B) CglThrB-WT with magnesium (purple sphere) and three phosphate/sulfate bound in the active site. The omit map of all three phosphates/sulfates is contoured to 3σ. (C) Superposition of both M. jannaschii and CglThrB-WT with both their magnesium (purple spheres) and l-isoleucine and ATP-γS from 1H74.