**7.1 Function of DAPE**

*Diaminopimelate epimerase* (DAPE, EC 5.1.1.7) catalyses the penultimate step in the lysine biosynthetic pathway whereby *L,L*-2,6-diaminopimelate (*LL*-DAP) is converted to *meso*-DAP (Fig. 1, Fig. 16). In *E. coli*, the enzyme is encoded by the *dap*F gene and is constitutively expressed (Neidhardt & Curtiss, 1996). DAPE was first characterised in 1957 using enzyme derived from crude extracts of *E. coli* (Work, 1957). The enzyme specifically recognises the *LL*-DAP isomer (Anita et al., 1957), whereas the *DD*-DAP isomer is not a substrate or inhibitor of the enzyme. Early studies noted that DAPE was inhibited by low concentrations of thiol-binding reagents and could be reactivated by reducing agents, suggesting the presence of an essential sulfhydryl group (Work, 1957). This finding was subsequently confirmed upon purification of DAPE to homogeneity (Wiseman, & Nichols, 1984).

Fig. 16. DAPE catalysed reaction.

DAPE catalyses the conversion of *LL*-DAP to *meso*-DAP by employing a "two-base" mechanism (Wiseman, & Nichols, 1984). The reaction involves two active site Cys residues, where the first Cys residue (73 in *H. influenzae*) acts as base abstracting proton from *LL*-DAP, while the second Cys residue (217 in *H. influenzae*) re-protonates the molecule to generate *meso*-DAP. The enzyme is also capable of catalysing the reverse reaction, with the two Cys residues reversing their roles (Wiseman, & Nichols, 1984).

#### **7.2 Structure of DAPE**

The structures of DAPE from four species have been described. These include DAPE from *B. anthracis* (PDB:2OTN), *H. influenza* (Cirilli et al., 1998; Lloyd et al., 2004)*,* and *M. tuberculosis* (Usha et al., 2009); and also the plant species *A. thaliana* (Pillai et al., 2009). The enzyme is a symmetrical monomer comprised of two domains containing eight β-strands and two αhelices (Cirilli et al., 1998) (Fig. 17).

Fig. 17. Structure of DAPE from *H. influenzae*. Domains are coloured pink and blue, active site cysteines (disulfide linked) are shown in yellow (PDB: 1BWZ).

This fold, first observed in *H. influenzae* DAPE, is now referred to as the DAP epimerase-like fold. The structure of DAPE from *H. influenzae* shows that each domain of the enzyme contributes one active site Cys (residues 73 and 217). The distal, non-reacting end of the substrate interacts via a number of hydrogen bonds to residues Asn157, Asp190, Arg209, Asn64, and Glu208 (Fig. 18). The nature of this interaction ensures that only the *LL*-DAP stereoisomer is recognised. Interestingly, DAPE adopts two distinct conformational states. In the absence of substrate, the enzyme exists in an open conformation, and upon binding substrate adopts a closed conformation (Pillai et al., 2007).

DAPE catalyses the conversion of *LL*-DAP to *meso*-DAP by employing a "two-base" mechanism (Wiseman, & Nichols, 1984). The reaction involves two active site Cys residues, where the first Cys residue (73 in *H. influenzae*) acts as base abstracting proton from *LL*-DAP, while the second Cys residue (217 in *H. influenzae*) re-protonates the molecule to generate *meso*-DAP. The enzyme is also capable of catalysing the reverse reaction, with the two Cys

The structures of DAPE from four species have been described. These include DAPE from *B. anthracis* (PDB:2OTN), *H. influenza* (Cirilli et al., 1998; Lloyd et al., 2004)*,* and *M. tuberculosis* (Usha et al., 2009); and also the plant species *A. thaliana* (Pillai et al., 2009). The enzyme is a symmetrical monomer comprised of two domains containing eight β-strands and two α-

Fig. 17. Structure of DAPE from *H. influenzae*. Domains are coloured pink and blue, active

This fold, first observed in *H. influenzae* DAPE, is now referred to as the DAP epimerase-like fold. The structure of DAPE from *H. influenzae* shows that each domain of the enzyme contributes one active site Cys (residues 73 and 217). The distal, non-reacting end of the substrate interacts via a number of hydrogen bonds to residues Asn157, Asp190, Arg209, Asn64, and Glu208 (Fig. 18). The nature of this interaction ensures that only the *LL*-DAP stereoisomer is recognised. Interestingly, DAPE adopts two distinct conformational states. In the absence of substrate, the enzyme exists in an open conformation, and upon binding

site cysteines (disulfide linked) are shown in yellow (PDB: 1BWZ).

substrate adopts a closed conformation (Pillai et al., 2007).

Fig. 16. DAPE catalysed reaction.

**7.2 Structure of DAPE** 

helices (Cirilli et al., 1998) (Fig. 17).

residues reversing their roles (Wiseman, & Nichols, 1984).

Fig. 18. Catalytic site of DAPE from *H. influenzae*. Hydrogen bond interactions (black dotted lines) at the distal site of the substrate analogue *LL*-AziDAP (arrow indicating position of the analogue) (PDB: 2GKE).
