Understanding Catalytic Specificity in Alanine Racemase from Quantum Mechanical and Molecular Mechanical Simulations of the Arginine 219 Mutant

Rubinstein, Amir; Major, Dan Thomas

doi:10.1021/bi1002629

Cited by 15 publications

(24 citation statements)

References 62 publications

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“…In search of Alr inhibitors, Mobashery and Johnston 60À62 prepared and investigated the reactions of the peptidyl (24)(25)(26) and amino acid (27,28) (Figure 17) C 10 -esters of deacetylcephalothin with purified enzymes in vitro. Compound 27 showed timedependent inactivation of E. coli Alr in a process that requires O-lactamase for the initial liberation of b-chloro-L-alanine from the cephalosporin.…”

Section: Inhibitors Of Alanine Racemasementioning

confidence: 99%

“…Reprotonation on the opposite side of the a-carbon carbanion produces the opposite enantiomer of the alanine substrate whereas reprotonation on the original side of the carbanion by the protonated Lys39 "-amino group yields the same enantiomer of alanine as that of the substrate. There are several reports describing either the role of Lys39, Arg219, and Tyr265 0 residues 24,[26][27][28] in the enzyme catalytic steps or substrate analogs used in structural studies to date 20,[29][30][31][32][33][34] . The structures of Alr with the covalent inhibitors alanine phosphonate and D-cycloserine showed that the inhibition of Alr was due to the formation of a stable covalent linkage with PLP 22,35 .…”

Section: Introductionmentioning

confidence: 99%

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Inhibitors of alanine racemase enzyme: a review

Azam

Jayaram

2015

Journal of Enzyme Inhibition and Medicinal Chemistry

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Alanine racemase is a fold type III PLP-dependent amino acid racemase enzyme catalysing the conversion of l-alanine to d-alanine utilised by bacterial cell wall for peptidoglycan synthesis. As there are no known homologs in humans, it is considered as an excellent antibacterial drug target. The standard inhibitors of this enzyme include O-carbamyl-d-serine, d-cycloserine, chlorovinyl glycine, alaphosphin, etc. d-Cycloserine is indicated for pulmonary and extra pulmonary tuberculosis but therapeutic use of drug is limited due to its severe toxic effects. Toxic effects due to off-target affinities of cycloserine and other substrate analogs have prompted new research efforts to identify alanine racemase inhibitors that are not substrate analogs. In this review, an updated status of known inhibitors of alanine racemase enzyme has been provided which will serve as a rich source of structural information and will be helpful in generating selective and potent inhibitor of alanine racemase.

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Section: Inhibitors Of Alanine Racemasementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Inhibitors of alanine racemase enzyme: a review

Azam

Jayaram

2015

Journal of Enzyme Inhibition and Medicinal Chemistry

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“…In an effort to understand the role of protonation of the pyridine nitrogen in PLP, we also studied the Arg219Glu mutant [66]. In this mutant, the pyridine nitrogen interacts with the Glu residue, necessitating a protonated state.…”

Section: Resultsmentioning

confidence: 99%

Molecular dynamics simulations of the intramolecular proton transfer and carbanion stabilization in the pyridoxal 5′-phosphate dependent enzymes l-dopa decarboxylase and alanine racemase

Lin

Gao

Rubinstein

et al. 2011

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Molecular dynamics simulations using a combined quantum mechanical and molecular mechanical (QM/MM) potential have been carried out to investigate the internal proton transfer equilibrium of the external aldimine species in l-dopa decarboxylase, and carbanion stabilization by the enzyme cofactor in the active site of alanine racemase. Solvent effects lower the free energy of the O-protonated PLP tautomer both in aqueous solution and in the active site, resulting a free energy difference of about – 1 kcal/mol relative to the N-protonated Schiff base in the enzyme. The external aldimine provides the dominant contribution to lowering the free energy barrier for the spontaneous decarboxylation of l-dopa in water, by a remarkable 16 kcal/mol, while the enzyme l-dopa decarboxylase further lowers the barrier by 8 kcal/mol. Kinetic isotope effects were also determined using a path integral free energy perturbation theory on the primary 13C and the secondary 2H substitutions. In the case of alanine racemase, if the pyridine ring is unprotonated as that in the active site, there is destabilizing contribution to the formation of the α-carbanion in the gas phase, although when the pyridine ring is protonated the contribution is stabilizing. In aqueous solution and in alanine racemase, the α-carbanion is stabilized both when the pyridine ring is protonated and unprotonated. The computational studies illustrated in this article show that combined QM/MM simulations can help provide a deeper understanding of the mechanisms of PLP-dependent enzymes. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.

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“…56 Therefore, the main catalytic effect of PLP in AlaR may be ascribed to solvation. [59][60][61] It has been assumed that in such an enzyme the non-protonated form generates an intermediate with a limited lifetime which aids the enzyme in avoiding side reactions. 62 Quantum mechanical and molecular dynamics simulations were recently carried out on the wild type and Arg219Glu mutant.…”

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confidence: 99%

Drug Discovery Targeting Amino Acid Racemases

Conti¹,

Tamborini²,

Pinto³

et al. 2011

Chem. Rev.

112

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Amino acids are among the most important molecules in nature since they play central roles both as building blocks of proteins and as intermediates in metabolism. The amino acid sequence dictates protein folding, the native three dimensional structure, and protein stability. Furthermore, the peculiar chemical properties of the amino acids forming the active site and their interplay determine protein function and regulation. All amino acids found in proteins, except glycine, possess a stereogenic center at the a-carbon atom. Millions of years of evolution have resulted in the virtually complete homochirality of such a stereogenic center, i.e. the L-enantiomer, in mammals. 1 This selection of the L-amino acids by nature is generally considered to be a result of chance. 2 Since the cornerstone of the protein-ligand recognition is the multi-point attachment theory, it turns out that the configuration of the a-carbon atom of amino acids strongly affects the protein-ligand interaction. Nevertheless, during the last half of the twentieth century, various studies evidenced the presence of Damino acids in some plants and bacteria. 3,4 These compounds were either found in a free state or in peptides and proteins. Most bacteria produce significant amounts of D-alanine (D-Ala) and D-glutamate (D-Glu), which are incorporated into peptidoglycan. 5 Peptidoglycan is a strong and elastic polymer of the bacterial wall, which is capable to counteract the osmotic pressure of the cell, maintaining cell shape and anchoring components of the cell envelope. 6 The number of D-amino acids present in the structure of peptidoglycan seems to constitute a measure of protection against peptidase and protease attacks. So far, no peptidase capable of hydrolyzing a peptide bond characterized by the sequence D-D or D-L amino acids has been isolated in mammals. In addition, several antibiotics produced by prokaryotes (e.g. bacitracin, actinomycin D) contain D-amino acids (Figure 1). It has been recently demonstrated that bacteria synthesize a pool of different D-amino acids, including D-methionine (D-Met) and D-leucine (D-Leu) in Vibrio cholerae and D-tyrosine (D-Tyr) and D-phenylalanine (D-Phe) in Bacillus subtilis. By selectively incorporating them in the peptidoglycan cell wall, bacteria cope to different environmental stresses. 7

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Understanding Catalytic Specificity in Alanine Racemase from Quantum Mechanical and Molecular Mechanical Simulations of the Arginine 219 Mutant

Cited by 15 publications

References 62 publications

Inhibitors of alanine racemase enzyme: a review

Inhibitors of alanine racemase enzyme: a review

Molecular dynamics simulations of the intramolecular proton transfer and carbanion stabilization in the pyridoxal 5′-phosphate dependent enzymes l-dopa decarboxylase and alanine racemase

Drug Discovery Targeting Amino Acid Racemases

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