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Dihydropteroate Synthase

Lyra Hall '14 and Rina Petek '15


I. Introduction

Dihydropteroate synthase (DHPS) is an enzyme involved in the Bacillus anthracis folate synthesis pathway. The enzyme has two binding pockets: one which binds dihydropterin pyrophosphate (DHPP) and one which binds p-amino benzoic acid(pABA). DHPS catalyzes the reaction which produces 7,8-dihydropteroate from these two substrates.

The folate synthesis pathway is a crucial pathway for synthesizing amino acids. Obviously, without amino acids, bacteria cannot function, so this is an efficient method to inhibit bacterial growth. In the past, the pABA pocket of DPHS has been targeted using sulfonamides. However, due to high clinical usage of sulfonamides, bacterial populations have built up resistance to this method. The most logical way to combat this bacterial resistance is to simply target the enzyme's other binding site, called the pterin pocket.

Pterins are defined as a class of heterocyclic compounds which mimic dihydropterin pyrophosphate (DHPP): one of the enzyme's two natural substrates. The key pharmacophore elements of DHPP are the two nitrogenous aromatic rings with an amino group and a carbonyl group para to each other.

As a point of interest, the first molcule discovered to inhibit DHPS by binding to the pterin binding site was 6-(methylamino)-5-nitroisocytosine known as MANIC.

II. General Structure

Dihydropteroate synthase is made up of two identical monomers of a classic TIM barrel structure, which signifies 8 alpha helices alternating with 8 beta sheets. With the assistance of flexible linker strands, these helices and sheets curve in on each other to make a shape called a toroid, or doughnut.The beta sheets form the inner wall of the doughnut while the alpha helices form the outer wall. The core of the doughnut contains no peptide backbone, but is filled with side chains of hydrophobic amino acids.

III. Structure of Pterin Pocket

The pterin binding site is located in the TIM barrel.The key residues that recognize the pterin substrate (in our model, PtPP) through hydrogen bonding are Asn120, Asp184, Lys220, and a water molecule. Arg254 and His 256 hydrogen bond with the beta phosphate on PtPP.

Interestingly, two flexible loops are highly conserved throughout DHPS analogs of different bacterial species. (loop 1 and loop2) Loop 2 is not well characterized in our model, as the loops only become fully ordered in a dimer, which has yet to be crystallized. Loop 1 is hypothesized to provide a conserved aspartate to contribute to the catalytic mechanism of the pABA binding site. Arg 68 on Loop 2 is thought to stabilize the pterin site of the unbound enzyme due to the presence of a guanidinium group which structurally resembles the pterin ring.

IV. Structure of pABA Pocket

The pABA binding site is poorly characterized since it is made up mostly of difficult-to-characterize flexible loops. Nevertheless, Babaoglu et al managed to crystallize a novel DHPS-product analog complex and were able to infer some critical interactions that pABA makes in the barrel. The aromatic group of pABA has hydrophobic interactions with Lys220 and Phe189, and the amine group hydrogen bonds with Ser218.

V. Advantages and Disadvantages of Targeting The Pterin Site for Drug Design

There are several advantages to targeting the pterin site rather than the pABA site. First, all known mutations that confer resistance to sulfa drugs occur within the pABA binding pocket. Therefore, inhibitory compounds that bind to the pterin site can get around the most prevalent method of bacterial resistance. Second, the pABA site is composed of flexible loops, whereas the pterin site is severely constrained by its structural integrity to the TIM barrel. In other words, there is much less potential for bacterial mutation to the pterin site that successfully maintains proper function of the protein. Third, because of the tight restrictions placed by necessity upon it, the pterin site is highly conserved across many species of bacteria, so it presents an opportunity to create a drug that could potentially be effective for a wide variety of bacterial-caused illnesses.

However, there are some drawbacks. Pterin compounds tend to have poor solubility. Due to their planar structure, they are much more stable in a crystal lattice than they are in solution. Drugs must dissolve into the body to be effective, so historically compounds with poor solubility have been found to not make good drugs. Hevener et al say this problem can potentially be addressed by adding anionic functional groups. A second potential drawback to this pterin-based method of design is that other opportunities may be missed out on. In the field of drug design, a receptor-based approach to design is preferred. A potential solution to this issue has already been identified: one of the compounds  Hevener et al discovered through their screening process is not a pterin compound, but successfully binds the pterin pocket. Following this line of inquiry may lead to a much more effective drug.  

VI. References

Hevener, K.E., Yun, M., Qi, J., Kerr, I.D., Babaoglu, K., Hurdle, J.G., Balakrishna, K., White, S. W., and Lee, R.E. 2010. Structural Studies of Pterin-Based Inhibitors of Dihyropteroate Synthase. J Med Chem. 53(1): 166-177.

Babaoglu, K., Qi, J., Lee, R.E., and White, S.W. Crystal Structure of 7,8-Dihydropteroate Synthase from Bacillus anthracis: Mechanism and Novel Inhibitor Design. Structure, Vol. 12, 1705–1717.

Adapted from the Wikimedia Commons file "File:Guanidine-2D.png"

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