| 1. |
- Bernardo-Garcia, Noelia, et al.
(author)
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Cold-induced aldimine bond cleavage by Tris in Bacillus subtilis alanine racemase
- 2019
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In: Organic and biomolecular chemistry. - : Royal Society of Chemistry. - 1477-0520 .- 1477-0539. ; 17:17, s. 4350-4358
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Journal article (peer-reviewed)abstract
- Pyridoxal 5'-phosphate (PLP) is a versatile cofactor involved in a large variety of enzymatic processes. Most of PLP-catalysed reactions, such as those of alanine racemases (AlaRs), present a common resting state in which the PLP is covalently bound to an active-site lysine to form an internal aldimine. The crystal structure of BsAlaR grown in the presence of Tris lacks this covalent linkage and the PLP cofactor appears deformylated. However, loss of activity in a Tris buffer only occurred after the solution was frozen prior to carrying out the enzymatic assay. This evidence strongly suggests that Tris can access the active site at subzero temperatures and behave as an alternate racemase substrate leading to mechanism-based enzyme inactivation, a hypothesis that is supported by additional X-ray structures and theoretical results from QM/ MM calculations. Taken together, our findings highlight a possibly underappreciated role for a common buffer component widely used in biochemical and biophysical experiments.
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| 2. |
- Bernardo-Garcia, Noelia, et al.
(author)
-
Structural Bioinformatics in Broad-Spectrum Racemases : a new path in anti-microbial research
- 2016
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In: Current organic chemistry. - : Bentham Science. - 1385-2728 .- 1875-5348. ; 20:11, s. 1222-1231
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Journal article (peer-reviewed)abstract
- D-amino acids are essential components of the bacterial cell wall and play notable roles in microbiology as regulators, for example in sporulation, biofilm formation or interspecies communication. Racemases are the specific enzymes catalyzing the interconversion of L-amino acids to D-amino acids. While most of racemases are mono-specific, a family of broad-spectrum racemases that can racemize ten of the 19 natural chiral amino acids has been recently reported. These enzymes can interconvert radically different residues such as aliphatic and positively charged residues producing non-canonical D-amino acids. Crystal structures together with bioinformatics allowed identification of the residues defining the molecular footprint in broad-spectrum racemases, the specific features of their active sites and the structural basis of their promiscuity. Here we review the recent knowledge on this family compared with the well established of alanine racemases. This structural information is a prerequisite for the development of novel drugs against the important human pathogens for which broad-spectrum racemases play a key role.
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