**2.2.5 Allosteric site**

As described in Section 2.1.1, lysine is an allosteric modulator of DHDPS function, partially inhibiting DHDPS activity. The lysine binding site is situated in a crevice at the interface of the tight dimer, distal from the active site, but connected via a water filled channel (Fig. 3). The crystal structure of lysine-bound *E. coli* DHDPS shows two lysine molecules bound per dimer (four per tetramer) with each molecule interacting with both monomers and the adjacent lysine molecule (Blickling et al., 1997c) (Fig. 5).

Fig. 5. *E. coli* DHDPS allosteric site with two lysine (boxed - red) molecules bound (PDB: 2ATS).

As stated earlier, seven residues are believed to be involved in binding lysine; Ser48, Ala49, His53, His56, Asn80, Glu84 and Tyr106. All these residues show slightly altered conformations in the presence of lysine, moving to accommodate the molecule (Blickling et al., 1997a). Importantly, Tyr106 moves towards the carboxyl group of lysine, which alters the aromatic stacking of Tyr106 and Tyr107. Otherwise, very few changes are observed upon lysine binding, with no significant secondary structure or quaternary structure change occurring (Dobson et al., 2005a). Most of the residues identified as important in the lysine allosteric binding site are not conserved in those DHDPS enzymes that are not inhibited by lysine (Burgess et al., 2008; Kefala et al., 2008; Voss et al., 2010; Wolterink-van Loo et al., 2008).

#### **2.2.6 Alternative quaternary architecture**

Whilst the DHDPS monomer from most bacteria has a molecular mass of approximately 31 kDa, the plant enzymes are larger. For example, DHDPS from *Nicotiana sylvestris* (Blickling et al., 1997b) has a relative molecular mass of 36 kDa, whilst DHDPS from *Pivus sativum* (Dereppe et al., 1992) has been reported to be a homotrimer of 43 kDa monomers based on gel filtration liquid chromatography studies, although this result is uncorroborated. The only plant DHDPS structure solved to date is from *N. sylvestris* (Blickling et al., 1997b). As for the bacterial enzymes, it is a homotetramer, described as a dimer of dimers. The contact areas within the tight dimer are similar within the plant and bacterial enzyme, with 13 of the 19 residues contributing to the interface conserved in both bacteria and plants. However, as Figure 6 shows, the plant dimer of dimers has an alternative architecture, namely the residues involved at the weak dimer interface are located on the opposite face of the monomer. The plant enzyme can thus be described as a "back-to-back" arrangement of dimers

the tight dimer, distal from the active site, but connected via a water filled channel (Fig. 3). The crystal structure of lysine-bound *E. coli* DHDPS shows two lysine molecules bound per dimer (four per tetramer) with each molecule interacting with both monomers and the

Fig. 5. *E. coli* DHDPS allosteric site with two lysine (boxed - red) molecules bound (PDB: 2ATS).

As stated earlier, seven residues are believed to be involved in binding lysine; Ser48, Ala49, His53, His56, Asn80, Glu84 and Tyr106. All these residues show slightly altered conformations in the presence of lysine, moving to accommodate the molecule (Blickling et al., 1997a). Importantly, Tyr106 moves towards the carboxyl group of lysine, which alters the aromatic stacking of Tyr106 and Tyr107. Otherwise, very few changes are observed upon lysine binding, with no significant secondary structure or quaternary structure change occurring (Dobson et al., 2005a). Most of the residues identified as important in the lysine allosteric binding site are not conserved in those DHDPS enzymes that are not inhibited by lysine

(Burgess et al., 2008; Kefala et al., 2008; Voss et al., 2010; Wolterink-van Loo et al., 2008).

Whilst the DHDPS monomer from most bacteria has a molecular mass of approximately 31 kDa, the plant enzymes are larger. For example, DHDPS from *Nicotiana sylvestris* (Blickling et al., 1997b) has a relative molecular mass of 36 kDa, whilst DHDPS from *Pivus sativum* (Dereppe et al., 1992) has been reported to be a homotrimer of 43 kDa monomers based on gel filtration liquid chromatography studies, although this result is uncorroborated. The only plant DHDPS structure solved to date is from *N. sylvestris* (Blickling et al., 1997b). As for the bacterial enzymes, it is a homotetramer, described as a dimer of dimers. The contact areas within the tight dimer are similar within the plant and bacterial enzyme, with 13 of the 19 residues contributing to the interface conserved in both bacteria and plants. However, as Figure 6 shows, the plant dimer of dimers has an alternative architecture, namely the residues involved at the weak dimer interface are located on the opposite face of the monomer. The plant enzyme can thus be described as a "back-to-back" arrangement of dimers

**2.2.6 Alternative quaternary architecture** 

adjacent lysine molecule (Blickling et al., 1997c) (Fig. 5).

(Fig. 6) compared to the "head-to-head" arrangement observed for bacterial DHDPS (Fig. 3). Compared to the bacterial interface, the weak dimer interface of *N. sylvestris* DHDPS is larger than its bacterial counterpart, burying 810 Å2 surface area, which is reflected in the greater number of residues contributing to inter-subunit contacts. The additional residues of the Cterminus, as well as the novel quaternary structure of *N. sylvestris* DHDPS, reduces the central water filled cavity and results in a tetramer where all subunits are in contact with each other (Fig. 6). Despite the significant structural differences between the plant and bacterial enzymes, the position and orientation of all active site residues are conserved. Most strikingly, considering the rearrangement of dimers, lysine binds at an equivalent binding pocket at the interfaces of the two monomers of a dimer in both the *E. coli* (Blickling et al., 1997a) and *N. sylvestris* (Blickling et al., 1997b) enzymes. The lysine molecules also bind in the same orientation, with coordination of the α-amino and α-carboxyl groups almost identical.

Fig. 6. *N. sylvestris* DHDPS, showing an alternate architecture ("back-to-back" arrangement of dimers) to that observed for bacterial DHDPS enzymes.

In addition, other quaternary structures of bacterial DHDPS enzymes have been reported. For example, DHDPS from methicillin-resistant *S. aureus* (MR*SA*) has recently been shown to be a dimer in solution (Burgess et al., 2008), with enzymatic activity similar to that of the wild-type *E. coli* tetramer. Several DHDPS enzymes have also been identified in *Agrobacterium tumefaciens,* with two forms crystallising as dimers (3B4U, 2R8W) and one as a hexamer (2HMC), although the function of this enzyme has yet to be confirmed.

#### **2.3 Inhibition of DHDPS**

A number of potential DHDPS inhibitors have been synthesised and characterised. A variety of heterocyclic analogues of DHDP and HTPA have been shown to act as moderate inhibitors of DHDPS (Hutton et al., 2007). Analogues of the cyclic lactol form of ASA, including homoserine lactone and 2-aminocyclopentanone, show non-competitive moderate inhibition with *K*i = 12-24 mM (Hutton et al., 2007). Analogues of the straight chain hydrate form of ASA have also been investigated, with aspartic acid showing mixed type inhibition with *K*i = 90-140 µM (Hutton et al., 2007). Product analogues have also been investigated, exhibiting moderate DHDPS inhibition. More success was achieved with inhibitors based on the acyclic enzyme-bound DHDPS intermediates, such as diethyl (*E,E*)-4-oxo-2,5 heptadienedioate (Turner et al., 2005) and a bis-oxime ester (Boughton et al., 2008), which irreversibly inhibit DHDPS. Interestingly, several of these compounds have displayed clear differentiation in inhibition of DHDPS enzymes from different species (Mitsakos et al., 2008), suggesting the potential for targeting compounds to specific pathogens.
