**1. Introduction**

224 Biochemistry

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Lysine is an essential amino acid in the mammalian diet, but can be synthesised *de novo* in bacteria, plants and some fungi (Dogovski et al., 2009; Hutton et al., 2007). In bacteria, the lysine biosynthesis pathway, also known as the diaminopimelate (DAP) pathway (Fig. 1), yields the important metabolites *meso*-2,6-diaminopimelate (*meso*-DAP) and lysine. Lysine is utilised for protein synthesis in bacteria and forms part of the peptidoglycan cross-link structure in the cell wall of most Gram-positive species; whilst *meso*-DAP is the peptidoglycan cross-linking moiety in the cell wall of Gram-negative bacteria and also Gram-positive *Bacillus* species (Burgess et al., 2008; Mitsakos et al., 2008; Voss et al., 2010) (Fig. 1).

The synthesis of *meso*-DAP and lysine begins with the condensation of pyruvate (PYR) and *L*-aspartate-semialdehyde (ASA) by the enzyme *dihydrodipicolinate synthase* (DHDPS, EC 4.2.1.52) (Blickling et al., 1997a; Mirwaldt et al., 1995; Voss et al., 2010; Yugari & Gilvarg, 1965). The product of the DHDPS-catalysed reaction is an unstable heterocycle, 4-hydroxy-2,3,4,5-tetrahydro-*L,L*-dipicolinic acid (HTPA) (Fig. 1). HTPA is non-enzymatically dehydrated to produce dihydrodipicolinate (DHDP), which is subsequently reduced by the NAD(P)H-dependent enzyme, *dihydrodipicolinate reductase* (DHDPR, EC 1.3.1.26), to form *L*-2,3,4,5,-tetrahydrodipicolinate (THDP) (Dommaraju et al., 2011; Girish et al., 2011; Reddy et al., 1995, 1996) (Fig. 1). The metabolic pathway then diverges into four sub-pathways depending on the species, namely the succinylase, acetylase, dehydrogenase and aminotransferase pathways (Dogovski et al., 2009; Hutton et al., 2007) (Fig. 1).

The most common of the alternative metabolic routes is the succinylase pathway, which is inherent to many bacterial species including *Escherichia coli*. This sub-pathway begins with the conversion of THDP to N-succinyl-*L*-2-amino-6-ketopimelate (NSAKP) catalysed by *2,3,4,5-tetrahydropyridine-2-carboxylate N-succinyltransferase* (THPC-NST, EC 2.3.1.117).

<sup>\*</sup> Sarah. C. Atkinson1, Sudhir R. Dommaraju1, Matthew Downton2, Lilian Hor1, Stephen Moore2, Jason J. Paxman1, Martin G. Peverelli1, Theresa W. Qiu1, Matthias Reumann2, Tanzeela Siddiqui1, Nicole L. Taylor1, John Wagner2, Jacinta M. Wubben1 and Matthew A. Perugini1,3

*<sup>1</sup>Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia* 

*<sup>2</sup>IBM Research Collaboratory for Life Sciences-Melbourne, Victorian Life Sciences Computation Initiative, University of Melbourne, Parkville, Victoria, Australia* 

*<sup>3</sup>Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia*

NSAKP is then converted to N-succinyl-*L,L*-2,6,-diaminopimelate (NSDAP) by *Nsuccinyldiaminopimelate aminotransferase* (NSDAP-AT, EC 2.6.1.17), which is subsequently desuccinylated by *succinyldiaminopimelate desuccinylase* (SDAP-DS, EC 3.5.1.18) to form *L,L*-2,6-diaminopimelate (*LL*-DAP) (Kindler & Gilvarg., 1960; Ledwidge & Blanchard., 1999; Simms et al., 1984) (Fig. 1). *LL*-DAP is then converted to *meso*-DAP by the enzyme diaminopimelate epimerase (DAPE, EC 5.1.1.7) (Wiseman, & Nichols, 1984) (Fig. 1).

Fig. 1. Diaminopimelate pathway in bacteria.

As for the succinylase pathway, the acetylase pathway involves four enzymatic steps, but incorporates N-acetyl groups rather than N-succinyl moieties. This pathway is common to several *Bacillus* species, including *B. subtilis* and the anthrax-causing pathogen *B. anthracis* (Chatterjee & White., 1982; Peterkofsky & Gilvarg., 1961; Sundharadas & Gilvarg., 1967). The sub-pathway begins with the conversion of THDP to N-acetyl-(*S*)-2-amino-6-ketopimelate (NAAKP) catalysed by *tetrahydrodipicolinate N-acetyltransferase* (THDP-NAT, EC 2.3.1.89), followed by conversion to N-acetyl-(2*S*)-2,6,-diaminopimelate (NADAP) by *aminotransferase A* (ATA, EC 2.6.1). NADAP is subsequently deacetylated to form DAP by the enzyme *Nacetyldiaminopimelate deacetylase* (NAD-DAC, EC 3.5.1.47) (Fig. 1). As in the succinylase pathway, *LL*-DAP is then converted to *meso*-DAP by DAPE (Fig. 1).

NSAKP is then converted to N-succinyl-*L,L*-2,6,-diaminopimelate (NSDAP) by *Nsuccinyldiaminopimelate aminotransferase* (NSDAP-AT, EC 2.6.1.17), which is subsequently desuccinylated by *succinyldiaminopimelate desuccinylase* (SDAP-DS, EC 3.5.1.18) to form *L,L*-2,6-diaminopimelate (*LL*-DAP) (Kindler & Gilvarg., 1960; Ledwidge & Blanchard., 1999; Simms et al., 1984) (Fig. 1). *LL*-DAP is then converted to *meso*-DAP by the enzyme

As for the succinylase pathway, the acetylase pathway involves four enzymatic steps, but incorporates N-acetyl groups rather than N-succinyl moieties. This pathway is common to several *Bacillus* species, including *B. subtilis* and the anthrax-causing pathogen *B. anthracis* (Chatterjee & White., 1982; Peterkofsky & Gilvarg., 1961; Sundharadas & Gilvarg., 1967). The sub-pathway begins with the conversion of THDP to N-acetyl-(*S*)-2-amino-6-ketopimelate (NAAKP) catalysed by *tetrahydrodipicolinate N-acetyltransferase* (THDP-NAT, EC 2.3.1.89), followed by conversion to N-acetyl-(2*S*)-2,6,-diaminopimelate (NADAP) by *aminotransferase A* (ATA, EC 2.6.1). NADAP is subsequently deacetylated to form DAP by the enzyme *Nacetyldiaminopimelate deacetylase* (NAD-DAC, EC 3.5.1.47) (Fig. 1). As in the succinylase

diaminopimelate epimerase (DAPE, EC 5.1.1.7) (Wiseman, & Nichols, 1984) (Fig. 1).

Fig. 1. Diaminopimelate pathway in bacteria.

pathway, *LL*-DAP is then converted to *meso*-DAP by DAPE (Fig. 1).

There are also two additional sub-pathways that are less common to bacteria. The aminotransferase pathway, catalysed by the enzyme *diaminopimelate aminotransferase* (*LL*-DAP-AT, EC 2.6.1.83), is found in plant, eubacterial and archaeal species (Hudson et al., 2006). This sub-pathway involves the conversion of the acyclic form of THDP, 1,2-amino-6 ketopimelate (AKP), to *meso*-DAP in a single step. *LL*-DAP is then converted in the second step of the sub-pathway to *meso*-DAP by DAPE, as for the acetylase and succinylase pathways (Fig. 1). The dehydrogenase pathway, which is common to *Corynebacterium* and some *Bacillus* species, converts THDP to *meso*-DAP, also in a single step (Misono et al., 1976). This sub-pathway employs the NADPH-dependent enzyme, *diaminopimelate dehydrogenase* (DAPDH, EC 1.4.1.16), which also employs AKP as the substrate (Fig. 1).

All four alternative pathways then converge to utilise the same enzyme for the final step of lysine biosynthesis, namely diaminopimelate decarboxylase (DAPDC, EC 4.1.1.20) (Ray et al., 2002). DAPDC catalyses the decarboxylation of *meso*-DAP to yield lysine and carbon dioxide. This step is important for the overall regulation of the lysine biosynthesis pathway since the downstream product, lysine, has been shown to allosterically inhibit DHDPS from plants and Gram-negative bacteria (Section 2.1.1, Fig. 1). DHDPS is therefore considered the rate-limiting enzyme of the pathway.

This book chapter will describe the function, structure, and regulation of the key enzymes functioning in the lysine biosynthesis pathway. Furthermore, given that several of these enzymes are the products of essential bacterial genes that are not expressed in humans, the pathway is of interest to antibiotic discovery research (Dogovski et al., 2009; Hutton et al., 2007). Accordingly, the chapter will also review the current status of rational drug design initiatives targeting essential enzymes of the lysine biosynthesis pathway in pathogenic bacteria.
