**2.2 Structure of DHDPS**

### **2.2.1 Subunit and quaternary structure of DHDPS**

DHDPS from *B. anthracis* (Blagova et al., 2006; Voss et al., 2010), *E. coli* (Mirwaldt et al., 1995), *Mycobacterium tuberculosis* (Kefala et al., 2008), *Thermoanaerobacter tengcongensis* (Wolterink-van Loo et al., 2008), *Thermotoga maritima* (Pearce et al., 2006), and several other species is a homotetramer in both crystal structure and solution (Fig. 3). In *E. coli*, the monomer is 292 amino acids in length and is composed of two domains (Mirwaldt et al., 1995). The N-terminal domain is a (β/α)8 TIM-barrel (residues 1-224) with the active site located within the centre of the barrel (Fig. 3). The C-terminal domain (residues 225-292) consists of three -helices and contains several key residues that mediate tetramerisation (Dobson et al., 2005a). The association of the four monomers leaves a large water-filled cavity in the centre of the tetramer, such that each monomer has contacts with two neighbouring monomers only. The tetramer can also be described as a dimer of dimers, with strong interactions between the monomers A & B and C & D at the so-called tight dimer interface, and weaker interactions between the dimers A-B and C-D at the weak dimer interface (Dobson et al., 2005a) (Fig. 3).

Fig. 3. *E. coli* DHDPS structure . The active sites, allosteric sites, dimerisation interface (tight dimer interface) and tetramerisation interface (weak dimer interface) are shown (PDB: 1YXC).

### **2.2.2 Active site**

The active site is located in cavities formed by the two monomers of the dimer. A long solventaccessible catalytic crevice with a depth of 10 Å is formed between -strands 4 and 5 of the barrel (Mirwaldt et al., 1995). Lys161, involved in Schiff-base formation is situated in the βbarrel near the catalytic triad of three residues, namely Tyr133, Thr44 and Tyr107, which act as a proton shuttle (Blickling et al., 1997a) (Fig. 4). Thr44 is hydrogen bonded to both Tyr133 and Tyr107 and its position in the hydrogen-bonding network may play a role in Schiff base formation and cyclisation (Dobson et al., 2005a). The dihedral angles of Tyr107 fall in the disallowed region of the Ramachandran plot, suggesting an important role in the enzyme's function (Mirwaldt et al., 1995). It is believed to be involved in shuttling protons between the active site and solvent (Dobson et al., 2005a). In contrast, Tyr133 plays an important role in substrate binding, donating a proton to the Schiff base hydroxyl. It is also thought to coordinate the attacking amino group of ASA, which requires the loss of a proton subsequent to cyclisation (Fig. 2). A marked reduction in activity is observed in single substitution mutants, highlighting the importance of this catalytic triad (Dobson et al., 2004a).

Situated at the entrance to the active site, Arg138 is essential for ASA binding (Dobson et al., 2005b). In the *E. coli* DHDPS structure, a hydrogen bond is formed between Arg138 and Tyr107 (Dobson et al., 2004a) and a water mediated hydrogen bond is formed between Arg138 and Tyr133 (Dobson et al., 2005a). Arg138 is thus also important for stabilisation of the catalytic triad, both of which are highly conserved in all DHDPS enzymes (Dobson et al., 2005a).

Fig. 3. *E. coli* DHDPS structure . The active sites, allosteric sites, dimerisation interface (tight dimer interface) and tetramerisation interface (weak dimer interface) are shown (PDB: 1YXC).

The active site is located in cavities formed by the two monomers of the dimer. A long solventaccessible catalytic crevice with a depth of 10 Å is formed between -strands 4 and 5 of the barrel (Mirwaldt et al., 1995). Lys161, involved in Schiff-base formation is situated in the βbarrel near the catalytic triad of three residues, namely Tyr133, Thr44 and Tyr107, which act as a proton shuttle (Blickling et al., 1997a) (Fig. 4). Thr44 is hydrogen bonded to both Tyr133 and Tyr107 and its position in the hydrogen-bonding network may play a role in Schiff base formation and cyclisation (Dobson et al., 2005a). The dihedral angles of Tyr107 fall in the disallowed region of the Ramachandran plot, suggesting an important role in the enzyme's function (Mirwaldt et al., 1995). It is believed to be involved in shuttling protons between the active site and solvent (Dobson et al., 2005a). In contrast, Tyr133 plays an important role in substrate binding, donating a proton to the Schiff base hydroxyl. It is also thought to coordinate the attacking amino group of ASA, which requires the loss of a proton subsequent to cyclisation (Fig. 2). A marked reduction in activity is observed in single substitution

mutants, highlighting the importance of this catalytic triad (Dobson et al., 2004a).

Situated at the entrance to the active site, Arg138 is essential for ASA binding (Dobson et al., 2005b). In the *E. coli* DHDPS structure, a hydrogen bond is formed between Arg138 and Tyr107 (Dobson et al., 2004a) and a water mediated hydrogen bond is formed between Arg138 and Tyr133 (Dobson et al., 2005a). Arg138 is thus also important for stabilisation of the catalytic triad, both of which are highly conserved in all DHDPS

**2.2.2 Active site** 

enzymes (Dobson et al., 2005a).

Fig. 4. *E. coli* DHDPS active site, illustrating the catalytic triad Thr44, Tyr133 and Tyr107 interdigitating from the opposing monomer (PDB: 1YXC).

## **2.2.3 Tight dimer interface**

In *E. coli* DHDPS, 1400 Å2 of surface area from one monomer in each dimer is buried at the tight dimer interface (Dobson et al., 2005a) (Fig. 3). This interface is made up of 25 residues from each monomer, with hydrogen bonds formed between Ser111 and Cys141, and hydrophobic interactions between Leu51 and Ala81, among others. In addition, Tyr107 of one monomer is coordinated with Tyr106 from the adjacent subunit, interdigitating across the monomer-monomer interface and thus forming a stabilising hydrophobic, sandwich-like stacking of aromatic rings.
