**4.3 Succinyldiaminopimelate desuccinylase**

*Succinyldiaminopimelate desuccinylase* (SDAP-DS, EC 3.5.1.18) catalyses the hydrolysis of Nsuccinyl-*L,L*-2,6,-diaminopimelate (NSDAP) to yield *L,L*-2,6-diaminopimelate (DAP) and succinate (Kindler & Gilvarg., 1960) (Fig. 1). Kinetic parameters for SDAP-DS from several bacterial species have been reported, with substrate *K*M and *k*cat values ranging from 0.73 - 1.3 mM and 140 - 200 s-1, respectively (Bienvenue et al., 2003; Born et al., 1998; Lin et al., 1988).

The gene encoding SDAP-DS, *dap*E*,* is present in a large number of bacterial species including, *C. glutamicum* (Wehrmann et al., 1994), *E. coli* (Bouvier et al., 1992), *Haemophilus influenzae,* (Born et al., 1998) and *Salmonella enterica* (Broder & Miller., 2003). In general, SDAP-DS contains approximately 375 residues and shares greater than 22% sequence identity across bacterial species. Alignment of SDAP-DS amino acid sequences show conservation of histidine and glutamate metal binding residues that are characteristic of metal-dependent amidases (Born et al., 1998).

Consistent with the conservation of metal binding residues, the activity of SDAP-DS enzymes are dependent on Zn2+ ions (Born et al., 1998; Lin *et al*., 1988). Futhermore, studies involving Zn K-edge extended X-ray absorption fine structure (EXAFS) analyses of *H. influenzae* SDAP-DS indicate that the enzyme contains dinuclear Zn2+ active sites (Cosper et al., 2003). Studies of *H. influenzae* SDAP-DS mutants by kinetics, electronic absorption spectroscopy and electron paramagnetic resonance spectroscopy showed that His67 and His349 coordinate Zn2+ ions, with His67 functioning in catalysis (Gillner et al., 2009). A similar study showed that residue Glu134 is also involved in catalysis, possibly functioning as an acid/base (Davis et al., 2006).

The crystal structure of zinc bound SDAP-DS has been determined (Fig. 13). Studies have shown that the enzyme forms a homodimer, with each monomer subunit containing a catalytic domain and a dimerisation domain (Nocek et al., 2010). The core of the catalytic domain is composed of an eight-stranded twisted β-sheet that is sandwiched between seven α-helices. The dimerisation domain adopts a two layer α+β sandwich fold and is comprised of a four stranded antiparallel β-sheet and two α-helices.

Fig. 13. Structure of dimeric *H. influenzae* SDAP-DS in complex with two zinc ions. the dimerisation (orange) and catalytic (blue) domains are indicated. Zinc ions (yellow) are bound by SDAP-DS active site residues (pink) (PDB: 3IC1).

The catalytic domain incorporates a negatively charged active site cleft, containing two zinc ions. One zinc ion is coordinated by the imidazole group and sidechain oxygens of His67 and Glu163, respectively, whilst another zinc ion is coordinated in a similar manner by His349 and Glu135. The zinc ions are bridged together by interaction with Asp100 and a water/hydroxide.

The availability of a structural model has resulted in a proposed mechanism for hydrolysis of NSDAP by SDAP-DS (Born et al., 1998; Nocek et al., 2010). It is hypothesised that NSDAP adopts an extended conformation when bound to the active site of the enzyme. The NSDAP amide carbonyl coordinates to an active site Zn2+ ion and becomes avaliable for nucleophilic attack. This binding event displaces a bridging water molecule, resulting in its hydrolysis by Glu134 and the generation of a zinc bound nucleophilic hydroxide. The hydroxide then attacks the target carbonyl carbon to form a η-1-μ-transition-state complex, which then resolves to release DAP and succinate.

The DAP isomers *L,L*-DAP and *D,L*-DAP are competitive inhibitors of *H. influenzae* SDAP-DS, exhibiting *K*i values of 8 and 12 mM, respectively (Born et al., 1998). Studies employing Zn K-edge EXAFS suggest that the *H. influenzae* SDAP-DS inhibitor, 5-mercaptopentanoic acid, may exert its effect through binding to active site Zn2+ ions (Cosper et al., 2003).
