Structural enzymology and inhibition of the bi-functional folate pathway enzyme HPPK-DHPS from the biowarfare agent Francisella tularensis

Abstract

Two valid targets for antibiotic development, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS), catalyze consecutive reactions in folate biosynthesis. In Francisella tularensis (Ft), these two activities are contained in a single protein, FtHPPK-DHPS. Although Pemble et al. (PLoS One 5, e14165) determined the structure of FtHPPK-DHPS, they were unable to measure the kinetic parameters of the enzyme. In this study, we elucidated the binding and inhibitory activities of two HPPK inhibitors (HP-18 and HP-26) against FtHPPK-DHPS, determined the structure of FtHPPK-DHPS in complex with HP-26, and measured the kinetic parameters for the dual enzymatic activities of FtHPPK-DHPS. The biochemical analyses showed that HP-18 and HP-26 have significant isozyme selectivity, and that FtHPPK-DHPS is unique in that the catalytic efficiency of its DHPS activity is only 1/260,000 of that of Escherichia coli DHPS. Sequence and structural analyses suggest that HP-26 is an excellent lead for developing therapeutic agents for tularemia, and that the very low DHPS activity is due, at least in part, to the lack of a key residue that interacts with the substrate p-aminobenzoic acid (pABA). A BLAST search of the genomes of ten F. tularensis strains indicated that the bacterium contains a single FtHPPK-DHPS. The marginal DHPS activity and the single copy existence of FtHPPK-DHPS in F. tularensis make this bacterium more vulnerable to DHPS inhibitors. Current sulfa drugs are ineffective against tularemia; new inhibitors targeting the unique pABA-binding pocket may be effective and less subject to resistance because any mutations introducing resistance may make the marginal DHPS activity unable to support the growth of F. tularensis.

Database: The coordinates and structure factors have been deposited in the Protein Data Bank under accession code 4PZV.

Keywords: DHPS; Francisella tularensis; HPPK; antibiotic; folate.

Figure 1

HPPK- and DHPS-catalyzed reactions and bisubstrate analogue inhibitors of HPPK. (A) The chemical structures of the substrates and products of HPPK- and DHPS-catalyzed reactions. (B) Chemical structure of HP-18 [16]. (C) Chemical structure of HP-26 [15].

Figure 2

K_d and IC₅₀ measurements. (A) Fluorometric titration of HPPK with HP-26. (B) Inhibition of HPPK by HP-26.

Figure 3

The FtHPPK-DHPS•HP-26 structure. (A) The polypeptide chain in the structure is shown as a ribbon diagram with helices (spirals) in cyan, strands (arrows) in orange, and loops (tubes) in grey. The DHPS module of the bifunctional enzyme is shaded. The HP-26 is shown as a stick model in the atomic color scheme (C in grey, N in blue, O in red, and S in yellow) outlined with the simulated annealing omit map (blue nets: F_o − F_c; contoured at 2.0 σ). (B) A zoomed-in view for the inhibitor and electron density.

Figure 4

Structure-based alignment of HPPK amino acid sequences. The amino acid numbering is that of FtHPPK-DHPS. The most conserved and highly conserved residues are shaded in black and gray, respectively.The active-site loops are indicated by horizontal bars. The residues involved in binding of HP and MgATP are marked below the sequence alignment with ▲ and ▼, respectively. Residues with 4.5 Å of both substrates are marked with ◆. Ft, F. tularensis; Ec, E. coli; Yp, Y. pestis; Ba, B. anthracis; Sa, S. aureus; Mt, M. tuberculosis; Sp, S. pneumoniae; Sc, S. cerevisiae.

Figure 5

Interactions between FtHPPK and HP-26. (A) Stereoview showing the superimposed FtHPPK•HP-26 (C atoms in cyan, this work) and FtHPPK•HP•MgAMPCPP (C atoms in magenta, PDB entry 3MCO) structures. (B) An enlarged view showing selected details of protein-ligand interactions in the two structures. Hydrogen bonds are indicated with dashed lines either in black (in FtHPPK•HP-26) or red (in FtHPPK•HP•MgAMPCPP). (B) Protein-inhibitor interactions as observed in the FtHPPK•HP-26 structure.

Figure 6

Stereoviews showing structural comparisons. (A) Superimposed FtHPPK•HP-26 (C atoms in cyan, this work) and EcHPPK•HP-26 (C atoms in orange, PDB entry 4F7V). Proteins are shown as Cα traces. HP-26 and selected side chains are shown as sticks. Hydrogen bonds are indicated with dashed lines either in black (in FtHPPK•HP-26) or red (in EcHPPK•HP-26). (B) Superimposed FtHPPK•HP-26 (C atoms in cyan, this work) and EcHPPK•HP-18 (C atoms in gray, PDB entry 3UDE). Hydrogen bonds are indicated with dashed lines either in black (in FtHPPK•HP-26) or red (in EcHPPK•HP-18).

Figure 7

Structure-based alignment of DHPS amino acid sequences. The amino acid numbering is that of FtHPPK-DHPS. The most conserved and highly conserved residues are shaded in black and gray, respectively. The active-site loops are indicated by horizontal bars. The residues involved in binding of HPPP and pABA are marked below the sequence alignment with ▲ and ▼, respectively. Residues within 4.5 Å of both substrates are marked with ◆. Sites of mutations that cause resistance to sulfa drugs are indicated by ● above the sequence alignment. Ft, F. tularensis; Mt2, M. tuberculosis DHPS 2; Ec, E. coli; Yp, Y. pestis; Ba, B. anthracis; Sa, S. aureus; Mt1, M. tuberculosis DHPS 1; Sp, S. pneumoniae; Sc, S. cerevisiae.

Figure 8

Steady-state kinetic analysis of FtHPPK-DHPS. (A) HPPK kinetics with MgATP varied while HP is fixed for determining the K_m for MgATP. (B) HPPK kinetics with HP varied while MgATP is fixed for determining the K_m for HP. (C) DHPS kinetics with pABA varied while HPPP is fixed.

Figure 9

Structural features in the DHPS of FtHPPK-DHPS. (A) Superimposed FtHPPK-DHPS•HP-26 (C atoms in cyan, this work) and YpDHPS•Mg²⁺•pABA•PP•XHP (C atoms in orange, PDB entry 3TYZ). Proteins are shown as ribbon diagrams. Selected ligands, including PP (pyrophosphate ion) and XHP [2–amino-6-metyhlidene-6,7-dihydropterindin-4(3H)-one], and side chains are shown as sticks. The hydrogen bond is indicated with a dashed line in red. (B) A zoomed-in view showing only the αloop7 in the two structures and the pABA in YpDHPS•Mg²⁺•pABA•PP•XHP. Indicated by a double-headed arrow is the distance between the S384 hydroxyl of FtHPPK-DHPS•HP-26 and the pABA of YpDHPS•Mg²⁺•pABA•PP•XHP.