A Molecular Dynamics Simulation of the Binding Modes of <scp>d</scp>-Glutamate and <scp>d</scp>-Glutamine to Glutamate Racemase

Puig, Eduard; Garcia‐Viloca, Mireia; González‐Lafont, Àngels; López, Inés López; Daura, Xavier; Lluch, José M.

doi:10.1021/ct049881g

Cited by 9 publications

(22 citation statements)

References 35 publications

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“…A solution of LLAziDAP (1-5 mg͞ml) was added in excess (10-100 eq) to a solution of DAP epimerase (0.54 mg͞ml) in a buffered solution (100 mM Tris⅐HCl͞1 mM EDTA͞1 mM DTT, pH 7.8) and incubated at room temperature for 16 h. To ensure complete inhibition, DAP epimerase activity was monitored spectrophotometrically by using a coupled assay with DAP dehydrogenase that detects the production of NADPH at 340 nm. An aliquot was removed and purified by RP-HPLC using a Jupiter (Torrance, CA) reverse-phase C 18 column. The purified enzyme was then dialyzed against Hepes buffer (pH 8.0, 25 mM Hepes͞5 mM DTT) and concentrated by using a Millipore Ultrafree Biomax 10K concentrator to a concen- …”

Section: Methodsmentioning

confidence: 99%

“…The x-ray crystal structure of glutamate racemase has been reported with the substrate analogue D-glutamine (3), but the lack of any strong interactions with the enzyme allows for a variety of possible binding modes in molecular modeling studies and precludes convincing mechanistic deductions (18). Recently, Rice and coworkers (19) have reported that the crystal structure of D-glutamate bound to glutamate racemase from Bacillus subtilis; this structure revealed a ''closed conformation'' for this enzyme upon ligand binding.…”

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confidence: 99%

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

Pillai

Cherney

Diaper

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

113

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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...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Pillai

Cherney

Diaper

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

113

View full text Add to dashboard Cite

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“…Two possibilities, which differ in the protonation states of the catalytic bases prior to the initial deprotonation, have been proposed (Glavas & Tanner, 2001; Ruzheinikov et al , 2005). Furthermore, stereochemistry, substrate ligation and active‐site protonation states have been investigated by molecular dynamics simulations (Möbitz & Bruice, 2004; Puig et al , 2005). Recently, the results from computational simulations on a quantum mechanical/molecular mechanical potential energy surface (Puig et al , 2006) indicated two possible roles for MurI as a catalyst and supported the mechanistic proposal by Rios et al (2000) for the PLP‐independent amino acid racemases.…”

Section: Side Pathwaysmentioning

confidence: 99%

Cytoplasmic steps of peptidoglycan biosynthesis

et al. 2008

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The biosynthesis of bacterial cell wall peptidoglycan is a complex process that involves enzyme reactions that take place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner side (synthesis of lipid-linked intermediates) and outer side (polymerization reactions) of the cytoplasmic membrane. This review deals with the cytoplasmic steps of peptidoglycan biosynthesis, which can be divided into four sets of reactions that lead to the syntheses of (1) UDP-N-acetylglucosamine from fructose 6-phosphate, (2) UDP-N-acetylmuramic acid from UDP-N-acetylglucosamine, (3) UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid and (4) D-glutamic acid and dipeptide D-alanyl-D-alanine. Recent data concerning the different enzymes involved are presented. Moreover, special attention is given to (1) the chemical and enzymatic synthesis of the nucleotide precursor substrates that are not commercially available and (2) the search for specific inhibitors that could act as antibacterial compounds.

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“…These features, along with similar strong hydrogen‐bonding networks at the substrate amino group, have been rationalized in the context of the difficult and remarkable chemical transformations performed by the enzyme given the solution state pKa values of cysteine (pKa ∼ 10) and the α‐carbon of glutamate (fully protonated form, pKa ∼ 21). Computational and physical chemistry measurements suggest the hydrophobic environment, the hydrogen‐bonding networks to form and stabilize the amino‐protonated, main chain cationic form of substrate, and the soft ionization energy of the thiolate base work in concert to minimize both the penalty of desolvation of the substrate upon binding and the large pKa differential between the substrate and enzyme at the transition state (Rios et al ., 2000; 2001; 2002; Puig et al ., 2005; 2006; 2007). Additional studies have implicated dynamics as a key element in overcoming the activation barrier for catalysis (Möbitz and Bruice, 2004).…”

Section: Biochemical and Structural Characterizationmentioning

confidence: 99%

Glutamate racemase as a target for drug discovery

Fisher¹

2008

Microbial Biotechnology

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SummaryThe bacterial cell wall is a highly cross‐linked polymeric structure consisting of repeating peptidoglycan units, each of which contains a novel pentapeptide substitution which is cross‐linked through transpeptidation. The incorporation of d‐glutamate as the second residue is strictly conserved across the bacterial kingdom. Glutamate racemase, a member of the cofactor‐independent, two‐thiol‐based family of amino acid racemases, has been implicated in the production and maintenance of sufficient d‐glutamate pool levels required for growth. The subject of over four decades of research, it is now evident that the enzyme is conserved and essential for growth across the bacterial kingdom and has a conserved overall topology and active site architecture; however, several different mechanisms of regulation have been observed. These traits have recently been targeted in the discovery of both narrow and broad spectrum inhibitors. This review outlines the biological history of this enzyme, the recent biochemical and structural characterization of isozymes from a wide range of species and developments in the identification of inhibitors that target the enzyme as possible therapeutic agents.

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A Molecular Dynamics Simulation of the Binding Modes of d-Glutamate and d-Glutamine to Glutamate Racemase

Cited by 9 publications

References 35 publications

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

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

Cytoplasmic steps of peptidoglycan biosynthesis

Glutamate racemase as a target for drug discovery

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