Structural insights into stereochemical inversion by diaminopimelate epimerase: An antibacterial drug target

Abstract

D-amino acids are much less common than their L-isomers but are widely distributed in most organisms. Many D-amino acids, including those necessary for bacterial cell wall formation, are synthesized from the corresponding L-isomers by ␣-amino acid racemases. The important class of pyridoxal phosphate-independent racemases function by an unusual mechanism whose details have been poorly understood. It has been proposed that the stereoinversion involves two active-site cysteine residues acting in concert as a base (thiolate) and an acid (thiol). Although crystallographic structures of several such enzymes are available, with the exception of the recent structures of glutamate racemase from Bacillus subtilis and of proline racemase from Trypanosoma cruzi, the structures either are of inactive forms (e.g., disulfide) or do not allow unambiguous modeling of the substrates in the active sites. Here, we present the crystal structures of diaminopimelate (DAP) epimerase from Haemophilus influenzae with two different isomers of the irreversible inhibitor and substrate mimic aziridino-DAP at 1.35-and 1.70-Å resolution. These structures permit a detailed description of this pyridoxal 5 -phosphate-independent amino acid racemase active site and delineate the electrostatic interactions that control the exquisite substrate selectivity of DAP epimerase. Moreover, the active site shows how deprotonation of the substrates' nonacidic hydrogen at the ␣-carbon (pKa Ϸ 29) by a seemingly weakly basic cysteine residue (pK a Ϸ 8 -10) is facilitated by interactions with two buried ␣-helices. Bacterial racemases, including glutamate racemase and DAP epimerase, are potential targets for the development of new agents effective against organisms resistant to conventional antibiotics.racemase ͉ stereochemical mechanism T he increase in microbial resistance to conventional antibiotics has rekindled intense interest in new methods of inhibiting bacterial cell wall biosynthesis (1), including blocking formation or utilization of D-amino acids such as D-alanine (2), D-glutamate (3), and meso-diaminopimelic acid (4) present in peptidoglycan (5). The inclusion of D-amino acids in the peptidoglycan layer of the cell wall is thought to provide bacteria with protection from the action of host proteases (2). Glutamate racemase (6, 7) and diaminopimelate (DAP) epimerase (EC 5.1.1.7) (8), as well as aspartate racemase (9) and proline racemase (10, 11), are examples of pyridoxal 5Ј-phosphate (PLP)-independent amino acid racemases that invert the configuration at the ␣-carbon of an amino acid without the use of cofactors, metals, or reducible keto or imino functionalities. The proposed ''two-base'' mechanism (11) for these enzymes involves one active-site cysteine thiolate acting as a base to deprotonate the ␣-carbon, while a second cysteine thiol functions as an acid to reprotonate the resulting planar carbanionic intermediate from the opposite face (Scheme 1). This process is not trivial, because the pK a of the ␣-proton is Ϸ21 for the fully protonated fo...

Dimerization of Bacterial Diaminopimelate Epimerase Is Essential for Catalysis

“…We therefore hypothesize that dimerization plays an important role in optimizing conformational dynamics critical for function. In DAP epimerase, the binding of ligand triggers a large conformational change, bringing the N-and C-terminal domains together (8,51). This event leads to the encapsulation of ligand and the reorganization of the active site with the formation of a network of hydrogen bonds at the catalytic center and the optimal positioning of the active site Cys residues ( Fig.…”

Superessential reactions in metabolic networks

“…DAP epimerase [enzyme commission number (EC) 5.1. 1.7], the final enzyme involved in the production of m-DAP, is actively being explored as a drug target (63)(64)(65)(66). Although DAP epimerase is essential for growth on all 54 carbon sources in E. coli, it is essential only in 54.2% of random viable networks, because many microbes can synthesize m-DAP through dia-minopimelate dehydrogenase (EC 1.4.1.16) (67).…”

Covalent Catalysis by Pyridoxal: Evaluation of the Effect of the Cofactor on the Carbon Acidity of Glycine

“…The first step in the mechanism for covalent catalysis by PLP is formation of an imine between the amino acid and PLP. Formation of this adduct labilizes all of the bonds of the α-imino carbon, because heterolytic bond cleavage with loss of H + , CO 2 or R + gives an enolate that is strongly stabilized by delocalization of negative charge onto the pyridine ring of the cofactor (Scheme 1).…”