Functional identification of the general acid and base in the dehydration step of indole-3-glycerol phosphate synthase catalysis

Abstract

The tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase is a proposed target for new antimicrobials and is a favored starting framework in enzyme engineering studies. Forty years ago, Parry proposed that the enzyme mechanism proceeds through two intermediates in a series of condensation, decarboxylation, and dehydration steps. X-ray crystal structures have suggested that Lys-110 (numbering according to the Sulfolobus solfataricus enzyme) behaves as a general acid both in the condensation and dehydration steps, but did not reveal an efficient pathway for the reprotonation of this critical residue. Our mutagenesis and kinetic experiments suggest an alternative mechanism whereby Lys-110 acts as a general acid in the condensation step, but another invariant residue, Lys-53, acts as the general acid in the dehydration step. These studies also indicate that the conserved residue Glu-51 acts as the general base in the dehydration step. The revised mechanism effectively divides the active site into discrete regions where the catalytic surfaces containing Lys-110 and Lys-53/Glu-51 catalyze the ring closure (i.e. condensation and decarboxylation) and dehydration steps, respectively. These results can be leveraged toward the development of novel inhibitors against this validated antimicrobial target and toward the rational engineering of the enzyme to produce indole derivatives that are highly prized by the pharmaceutical and agricultural industries.

Keywords: Bacterial Metabolism; Drug Development; Enzyme Kinetics; Enzyme Mechanisms; Protein Engineering; Tryptophan.

FIGURE 1.

The previously proposed chemical mechanism for the conversion of CdRP to IGP by IGPS has chemical inconsistencies, including the need to reprotonate Lys-110 following the first step.

FIGURE 2.

Solvent viscosity and isotope effects reveal the rate-determining step(s) in WT and variant ssIGPS enzymes. A, the kinetic mechanism of IGPS. There is a substantial SVE when the rate-determining step is diffusion-controlled, such as in product release, similar to what occurs at low temperatures (11). A substantial SDKIE is present only when the condensation step is rate-determining (see “Experimental Procedures” and supplemental “Experimental Procedures”). B, summary of the SVEs and SDKIEs for WT and variant ssIGPS enzymes at 75 °C indicates that single amino acid substitutions can change the rate-determining step and hence what k_cat reports on. A change in the rate-determining step also means that changes to k_cat may underreport the impact of amino acid substitutions on the individual kinetic steps.

FIGURE 3.

pH-rate profiles indicate that Glu-51 and Lys-53 act as the general base and acid, respectively, in the dehydration step of IGP production. Shown are the pH-rate profiles for k_cat at 25 °C (left) and 75 °C (right) for WT ssIGPS (A) and variants K53R (B) and E51Q (C). K_m values were pH-independent. Note the logarithmic scale on the y axes of these plots. In the K53R variant, the pK_a2 is substantially larger, as expected if the residue at position 53 acts as a general acid. The lack of the ascending limb in the pH-rate profile of the E51Q variant is consistent with Glu-51 acting as a general base in ssIGPS catalysis. Both the K53R and E51Q variants show shifts in their other pK_a values. These effects are likely caused by changes to the local microenvironment. For example, the loss of the negatively charged carboxylate group of Glu-51 would tend to lower the pK_a of the nearby side chain of Lys-53.

FIGURE 4.

The revised chemical mechanism reconciles structural, kinetic, and mutagenesis data for IGPS. A, assigned roles for the charged residues within the active site of ssIGPS. B, proposed revision to the chemical mechanism of ssIGPS, highlighting the roles of Lys-53 and Glu-51 during the dehydration step. Hydrogen extraction may occur from the alkyl hydrogen, as originally proposed (see Fig. 1), or may potentially occur from the amine hydrogen. C, the condensation/decarboxylation and dehydration steps occur in two different catalytic pockets. The first is shown in cyan and involves Lys-110 and Glu-159, and the second is shown in yellow and involves Lys-53 and Glu-51. The crystallographic docked structure of IGP is shown in the left panel. I2 was approximated using this docked structure through manual rotation within the ribose chain (right panel). These rotations show a plausible alignment of both the 2′-alcohol and the secondary amine of I2 with the Lys-53/Glu-51 catalytic surface. This rearrangement of the ribose chain can be obtained without considering thermal flexibility from either the phosphate anchor or the surrounding protein. D, surface rendering of the active site of ssIGPS docked with the reaction product IGP (Protein Data Bank code 1A53). The product is orientated with the indole ring facing inward toward to the core of the protein and the phosphate anchor located near the mouth of the cavity. The second catalytic surface is not located as deep within the pocket as the first. This positioning, along with the loss of the positive charge on Lys-53 following the dehydration, may aid in product release. Molecular graphics and analysis were performed using the UCSF Chimera package (25).