**8.1** *Diaminopimelate dehydrogenase*

*Diaminopimelate dehydrogenase* (DAPDH EC 1.4.1.16) is a NADPH dependant enzyme that catalyses the reductive amination of *L*-2-amino-6-ketopimelate (AKP), the acyclic form of *L*-2,3,4,5,-tetrahydrodipicolinate (THDP), to produce *meso*-DAP (Misono et al., 1976; Misono & Soda., 1980) (Fig. 1). It is assumed that the reaction occurs via an imine intermediate as a result of amination of *L*-2-amino-6-ketopimelate. Reduction of the imine by hydride transfer from NADPH generates *meso*-DAP (Scapin et al., 1998).

Only a small group of Gram-positive and Gram-negative bacteria posses DAPDH activity. These include *Bacillus sphaericus*, *Brevibacterium sp.*, *C. glutamicum* and *Proteus vulgaris* (Misono et al., 1979). Characterised DAPDH enzymes are comprised of approximately 320 residues and share greater than 27% sequence identity (Ishino et al., 1987; Hudson et al., 2011b). Kinetic studies of DAPDH from *C. glutamicum* has yielded *K*M values for NADPH, *L*-2-amino-6-ketopimelate and ammonia of 0.13 mM, 0.28 mM and 36 mM, respectively (Misono *et al*., 1986).

Some bacterial species possessing DAPDH activity use multiple pathways to synthesise lysine. For example, *C. glutamicium* (Schrumpf et al., 1991) can synthesise lysine by either the dehydrogenase or succinylase pathway, whilst *Bacillus macerans* (Hudson et al., 2011b) can employ enzymes of the dehydrogenase or acetylase pathways.

DAPDH from *C. glutamicum* forms a homodimer (Scapin et al., 1996) (Fig. 20). The DAPDH monomer subunit is comprised of (i) a dinucleotide binding domain, that is similar to but not identical to a classical Rossman fold, (ii) a dimerisation domain, and (iii) a C-terminal domain (Fig. 20). Monomer subunits interact via two α-helices and a three-stranded antiparallel β-sheet to form the dimer.

Fig. 20. Structure of dimeric *C. glutamicum* DAPDH in complex with NADPH and *L*-2-amino-6-methylene-pimelate. The dimerisation (orange), dinucleotide binding (blue), and C-terminal (green) domains are indicated. The cofactor NADPH (yellow) and inhibitor *L*-2-amino-6 methylene-pimelate (yellow) are bound by active site residues (pink) (PDB:1F06).

*Diaminopimelate dehydrogenase* (DAPDH EC 1.4.1.16) is a NADPH dependant enzyme that catalyses the reductive amination of *L*-2-amino-6-ketopimelate (AKP), the acyclic form of *L*-2,3,4,5,-tetrahydrodipicolinate (THDP), to produce *meso*-DAP (Misono et al., 1976; Misono & Soda., 1980) (Fig. 1). It is assumed that the reaction occurs via an imine intermediate as a result of amination of *L*-2-amino-6-ketopimelate. Reduction of the imine by hydride transfer

Only a small group of Gram-positive and Gram-negative bacteria posses DAPDH activity. These include *Bacillus sphaericus*, *Brevibacterium sp.*, *C. glutamicum* and *Proteus vulgaris* (Misono et al., 1979). Characterised DAPDH enzymes are comprised of approximately 320 residues and share greater than 27% sequence identity (Ishino et al., 1987; Hudson et al., 2011b). Kinetic studies of DAPDH from *C. glutamicum* has yielded *K*M values for NADPH, *L*-2-amino-6-ketopimelate and ammonia of 0.13 mM, 0.28 mM and 36 mM, respectively

Some bacterial species possessing DAPDH activity use multiple pathways to synthesise lysine. For example, *C. glutamicium* (Schrumpf et al., 1991) can synthesise lysine by either the dehydrogenase or succinylase pathway, whilst *Bacillus macerans* (Hudson et al., 2011b) can

DAPDH from *C. glutamicum* forms a homodimer (Scapin et al., 1996) (Fig. 20). The DAPDH monomer subunit is comprised of (i) a dinucleotide binding domain, that is similar to but not identical to a classical Rossman fold, (ii) a dimerisation domain, and (iii) a C-terminal domain (Fig. 20). Monomer subunits interact via two α-helices and a three-stranded

Fig. 20. Structure of dimeric *C. glutamicum* DAPDH in complex with NADPH and *L*-2-amino-6-methylene-pimelate. The dimerisation (orange), dinucleotide binding (blue), and C-terminal (green) domains are indicated. The cofactor NADPH (yellow) and inhibitor *L*-2-amino-6 methylene-pimelate (yellow) are bound by active site residues (pink) (PDB:1F06).

**8. Dehydrogenase pathway** 

(Misono *et al*., 1986).

**8.1** *Diaminopimelate dehydrogenase* 

antiparallel β-sheet to form the dimer.

from NADPH generates *meso*-DAP (Scapin et al., 1998).

employ enzymes of the dehydrogenase or acetylase pathways.

The crystal structure of the *C. glutamicum* DAPDH in complex with ligand shows that the oxidised cofactor, NADP+, is bound within each of the dinucleotide binding domains (Scapin et al., 1996). The domains exhibit open and closed conformations thought to represent the binding and active states of DAPDH, respectively (Scapin et al., 1996). In the closed conformation the NADP+ pyrophosphate forms seven additional noncovalent contacts. Subsequent studies demonstrate the product, *meso*-DAP, binds within an elongated cavity formed at the interface of the dimerisation and dinucleotide binding domains (Scapin et al., 1998).

Crystal structures of *C. glutamicum* complexed with the inhibitors (2*S*,5*S*)-2-amino-3-(3 carboxy-2-isoxazolin-5-yl)-propanoic acid (*K*i = 4.2 µM) and *L*-2-amino-6-methylenepimelate (*K*i = 5 µM) show that they form similar interactions with DAPDH as the product *meso*-DAP (Scapin et al., 1998). An additional hydrogen bond between the α-amino group of the *L*-2-amino-6-methylene-pimelate and the indole ring of DAPDH Trp144 is thought to account for the strong competitive inhibition observed (Scapin et al., 1998).
