Drug Discovery Targeting Amino Acid Racemases

Conti, Paola; Tamborini, Lucia; Pinto, Andrea; Blondel, Arnaud; Minóprio, Paola; Mozzarelli, Andrea; Micheli, Carlo De

doi:10.1021/cr2000702

Cited by 112 publications

(83 citation statements)

References 255 publications

(488 reference statements)

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“…Thus, whereas hydrolysis of alaphosphin releases L-Ala(P) (Fig. 1A), a competitive inhibitor for alanine racemase (14), enzymatic hydrolysis of dehydrophos will unmask 1-aminovinylphosphonate, ΔAla(P), in its monomethylated form (Fig. 1B).…”

mentioning

confidence: 99%

Revisiting the biosynthesis of dehydrophos reveals a tRNA-dependent pathway

Bougioukou

Mukherjee

Donk

2013

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

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Bioactive natural products containing a C-P bond act as mimics of phosphate esters and carboxylic acids, thereby competing with these compounds for active sites of enzymes. Dehydrophos (DHP), a broad-spectrum antibiotic, is a phosphonotripeptide produced by Streptomyces luridus , in which glycine and leucine are linked to an aminophosphonate analog of dehydroalanine, ΔAla(P). This unique feature, in combination with the monomethylation of the phosphonic acid, renders DHP a Trojan horse type antibiotic because peptidase-mediated hydrolysis will release methyl acetylphosphonate, a potent inhibitor of pyruvate dehydrogenase. Bioinformatic analysis of the biosynthetic gene cluster suggested that ΔAla(P) would be generated from Ser(P), the phosphonate analog of Ser, by phosphorylation and subsequent elimination, and that ΔAla(P) would be condensed with Leu-tRNA Leu . DhpH was anticipated to carry out this elimination/ligation cascade. DhpH is a multidomain protein, in which a pyridoxal phosphate binding domain is fused to an N -acetyltransferase domain related to the general control nonderepressible-5 (GCN5) family. In this work, the activity of DhpH was reconstituted in vitro. The enzyme was able to catalyze the β-elimination reaction of pSer(P) to generate ΔAla(P), but it was unable to condense ΔAla(P) with Leu. Instead, ΔAla(P) is hydrolyzed to acetyl phosphonate, which is converted to Ala(P) by a second pyridoxal phosphate-dependent enzyme, DhpD. Ala(P) is the substrate for the condensation with Leu-tRNA Leu catalyzed by the C-terminal domain of DhpH. DhpJ, a 2-oxoglutarate/Fe(II)-dependent enzyme, introduces the vinyl functionality into Leu-Ala(P) acting as a desaturase, and addition of Gly by DhpK in a Gly-tRNA Gly -dependent manner completes the in vitro biosynthesis of dehydrophos.

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confidence: 99%

Revisiting the biosynthesis of dehydrophos reveals a tRNA-dependent pathway

Bougioukou

Mukherjee

Donk

2013

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

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“…Amino acid racemases catalyze the interconversion of the L-and D-enantiomers using either a pyridoxal-5=-phosphate (PLP)-dependent or a PLP-independent mechanism. The PLP-dependent alanine racemase enzyme class has been studied extensively, owing to its potential as a target for antimicrobials (14). These enzymes are known to be necessary for D-alanine synthesis for peptidoglycan, as targeted disruption results in D-alanine auxotrophy (15).…”

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confidence: 99%

Amino Acid Racemization in Pseudomonas putida KT2440

Radkov

Moe

2013

J Bacteriol

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D-Amino acids have been shown to play an increasingly diverse role in bacterial physiology, yet much remains to be learned about their synthesis and catabolism. Here we used the model soil-and rhizosphere-dwelling organism Pseudomonas putida KT2440 to elaborate on the genomics and enzymology of D-amino acid metabolism. P. putida KT2440 catabolized the D-stereoisomers of lysine, phenylalanine, arginine, alanine, and hydroxyproline as the sole carbon and nitrogen sources. With the exception of phenylalanine, each of these amino acids was racemized by P. putida KT2440 enzymes. Three amino acid racemases were identified from a genomic screen, and the enzymes were further characterized in vitro. The putative biosynthetic alanine racemase Alr showed broad substrate specificity, exhibiting measurable racemase activity with 9 of the 19 chiral amino acids. Among these amino acids, activity was the highest with lysine, and the k cat /K m values with L-and D-lysine were 3 orders of magnitude greater than the k cat /K m values with L-and D-alanine. Conversely, the putative catabolic alanine racemase DadX showed narrow substrate specificity, clearly preferring only the alanine stereoisomers as the substrates. However, DadX did show 6-and 9-fold higher k cat /K m values than Alr with L-and D-alanine, respectively. The annotated proline racemase ProR of P. putida KT2440 showed negligible activity with either stereoisomer of the 19 chiral amino acids but exhibited strong epimerization activity with hydroxyproline as the substrate. Comparative genomic analysis revealed differences among pseudomonads with respect to alanine racemase genes that may point to different roles for these genes among closely related species.

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“…The first racemase described in 1951 was a bacterial alanine racemase (Alr). 87 Since then, a considerable number of similar enzymes have been reported in bacteria, archaea, and eukaryotes (reviewed in Yoshimura and Esak 91 and Conti et al 19 ).…”

Section: Production Of Non-canonical D-amino Acids: Bsrvmentioning

confidence: 99%

Peptidoglycan Remodeling by the Coordinated Action of Multispecific Enzymes

Álvarez

Espaillat

Hermoso

et al. 2014

Microbial Drug Resistance

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The peptidoglycan (PG) cell wall constitutes the main defense barrier of bacteria against environmental insults and acts as communication interface. The biochemistry of this macromolecule has been well characterized throughout the years but recent discoveries have unveiled its chemical plasticity under environmental stresses. Non-canonical D-amino acids (NCDAA) are produced and released to the extracellular media by diverse bacteria. Such molecules govern cell wall adaptation to challenging environments through their incorporation into the polymer, a widespread capability among bacteria that reveals the inherent catalytic plasticity of the enzymes involved in the cell wall metabolism. Here, we analyze the recent structural and biochemical characterization of Bsr, a new family of broad spectrum racemases able to generate a wide range of NCDAA. We also discuss the necessity of a coordinated action of PG multispecific enzymes to generate adequate levels of modification in the murein sacculus. Finally, we also highlight how this catalytic plasticity of NCDAA-incorporating enzymes has allowed the development of new revolutionary methodologies for the study of PG modes of growth and in vivo dynamics.

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Drug Discovery Targeting Amino Acid Racemases

Cited by 112 publications

References 255 publications

Revisiting the biosynthesis of dehydrophos reveals a tRNA-dependent pathway

Revisiting the biosynthesis of dehydrophos reveals a tRNA-dependent pathway

Amino Acid Racemization in Pseudomonas putida KT2440

Peptidoglycan Remodeling by the Coordinated Action of Multispecific Enzymes

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