Crystal structure and functional analysis of homocitrate synthase, an essential enzyme in lysine biosynthesis

Abstract

Homocitrate synthase (HCS) catalyzes the first and committed step in lysine biosynthesis in many fungi and certain Archaea and is a potential target for antifungal drugs. Here we report the crystal structure of the HCS apoenzyme from Schizosaccharomyces pombe and two distinct structures of the enzyme in complex with the substrate 2-oxoglutarate (2-OG). The structures reveal that HCS forms an intertwined homodimer stabilized by domain-swapping between the N- and C-terminal domains of each monomer. The N-terminal catalytic domain is composed of a TIM barrel fold in which 2-OG binds via hydrogen bonds and coordination to the active site divalent metal ion, whereas the C-terminal domain is composed of mixed alpha/beta topology. In the structures of the HCS apoenzyme and one of the 2-OG binary complexes, a lid motif from the C-terminal domain occludes the entrance to the active site of the neighboring monomer, whereas in the second 2-OG complex the lid is disordered, suggesting that it regulates substrate access to the active site through its apparent flexibility. Mutations of the active site residues involved in 2-OG binding or implicated in acid-base catalysis impair or abolish activity in vitro and in vivo. Together, these results yield new insights into the structure and catalytic mechanism of HCSs and furnish a platform for developing HCS-selective inhibitors.

FIGURE 1.

Schematic of the reaction catalyzed by homocitrate synthase.

FIGURE 2.

Crystal structure of S. pombe SpHCS and its overlay with M. tuberculosis α-IPMS. A, shown is a ribbon diagram of the SpHCS homodimer bound to 2-OG (closed lid motif) with the N-terminal domain, C-terminal subdomain I, and C-terminal subdomain II of monomer A depicted in red, orange, and yellow, respectively, and of monomer B depicted in green, blue, and violet, respectively. The Zn(II) atom is modeled as a dark gray sphere, and the bound 2-OG is rendered as sticks with yellow carbon atoms. B, shown is a ribbon diagram of the secondary structure of a monomer of SpHCS in complex with 2-OG with the secondary structure assigned following standard nomenclature for TIM barrel enzymes. The secondary structure is colored to distinguish between the N-terminal domain and C-terminal subdomains I and II. C, superimposition of a monomer of SpHCS (blue) in complex with 2-OG and a monomer of α-IPMS (gold) bound to 2-OIV. Coordination of the Zn(II) metal site in SpHCS and in α-IPMS is depicted as dashed orange and blue lines, respectively, whereas 2-OG and 2-OIV are depicted with yellow and magenta carbon atoms, respectively.

FIGURE 3.

SpHCS active site and 2-OG binding. A, shown is the active site of SpHCS (closed lid conformation; blue carbon atoms) in complex with 2-OG (yellow carbons) and Zn(II) (blue). Orange and yellow dashes represent coordination of the Zn(II) ion and hydrogen bonding to 2-OG, respectively. B, shown is the active site of α-IPMS (gold carbons) in complex with 2-OIV (magenta carbons) and Zn(II) (gold). The coordination of the Zn(II) ion and hydrogen bonds to 2-OIV are colored green and magenta, respectively. C, shown is the stereoview of the active site of the SpHCS 2-OG closed lid complex (rendered as in panel A) overlaid with the SpHCS apoenzyme (gray carbons). The Zn(II) ion and water molecules are represented as gray and red spheres, respectively. Electron density of the F_o − F_c simulated annealing omit map (contoured at 2.5 σ) corresponding to the 2-OG in the closed loop complex is depicted in cyan. D, shown is a ribbon diagram of a monomer of the SpHCS 2-OG closed lid complex (blue) superimposed with the corresponding monomer in the SpHCS apoenzyme (gray). The secondary structural elements that undergo conformational changes between the two structures are denoted.

FIGURE 4.

In vitro and in vivo activity of SpHCS. A and B, shown are Michaelis-Menten plots of the initial velocity versus AcCoA concentration (A) and 2-OG concentration (B) for the formation of homocitrate catalyzed by WT SpHCS. Data points are the average of triplicate measurements, and the error bars represent 1 S.D. C, in vivo yeast growth assays of LYS4 and LYS4 2-OG binding mutants.

FIGURE 5.

The lid motif of SpHCS. A, structural superimposition of the SpHCS·2-OG complexes with the open lid motif (monomer A is green, and monomer B is pale green) and the closed lid motif (monomer A is blue, and monomer B is cyan). The 2-OG and Zn(II) ion of the closed lid structure are depicted in yellow carbons and gray, respectively. B, shown is a stereoview of the active site of SpHCS·2-OG open lid complex (green carbon atoms with 2-OG rendered with orange carbons) overlaid with the SpHCS·2-OG closed lid complex (blue carbons). Electron density for the F_o − F_c-simulated annealing omit map (contoured at 2.0 σ) corresponding to the 2-OG in the open lid complex is shown in cyan. The orange and yellow dashes represent coordination of the Zn(II) ion and hydrogen bonding to 2-OG, respectively. C and D, shown are comparisons of the active site and lid motif of the SpHCS·2-OG open and closed lid complexes. Monomer A (blue carbons) and B (cyan carbons) of the closed lid complex are depicted with the Zn(II) ion (gray) and 2-OG (yellow carbons) (C), whereas monomer A (green carbons) and B (light green carbons) of the open lid complex are illustrated with the Co(II) ion (pink) and 2-OG (orange carbons) (D). Dashed lines denote the coordination of the metal ion (orange), hydrogen bonding to 2-OG (yellow), and hydrogen bonding within the lid motif (red). Residues in the lid motif that lack interpretable side-chain electron density are modeled as alanines, whereas the side chain of Tyr-332 in the open lid complex (D) is modeled with an occupancy of 0.5. E, shown is a model of a ternary complex of SpHCS bound to 2-OG (orange carbons) and AcCoA (violet carbons). The lid motif of the SpHCS·2-OG closed lid complex (cyan) is superimposed on the gray surface of the SpHCS·2-OG open lid complex.

FIGURE 6.

Catalytic mechanism and the SpHCS ternary complex model. A, shown is the proposed catalytic mechanism of HCS with 2-OG and AcCoA depicted as in Fig. 5E and homocitrate illustrated in green. B, shown is a stereoview of the active site of the modeled SpHCS ternary complex with 2-OG and the pantetheine arm of AcCoA rendered as in Fig. 5E. Dashed lines represent coordination of the Zn(II) ion (orange), hydrogen bonding to 2-OG (yellow), hydrogen bonding to the putative catalytic residues (blue), and the direction of the nucleophilic attack from the enolate tautomer of AcCoA to the C2 atom of 2-OG (red). C, in vivo yeast growth assays of the LYS4 mutants implicated in catalysis are shown.