Biology subjects

Dias, M. V. B.

Publications and source records attributed to Dias, M. V. B..

5 recordsLinked to original sources

bioRxiv2021

Mechanistic insights into dideoxygenation in gentamicin biosynthesis

Gentamicin is an important aminoglycoside antibiotic used for treatment of infections caused by Gram-negative bacteria. Although most of the biosynthetic pathway of gentamicin has been elucidated, a remaining intriguing question is how the intermediates JI-20A and JI-20B undergo a dideoxygenation to form gentamicin C complex. Here we show that the dideoxygenation process starts with GenP-catalyzed phosphorylation of JI-20A and JI-20Ba. The phosphorylated products are converted to C1a and C2a by concerted actions of two PLP (pyridoxal 5-phosphate)-dependent enzymes: elimination of water and then phosphate by GenB3 and double bond migration by GenB4. Each of these reactions liberates an imine which hydrolyses to a ketone or aldehyde and is then re-aminated by GenB3 using an amino donor. Crystal structures of GenB3 and GenB4 have guided site-directed mutagenesis to reveal crucial residues for the enzymes functions. We propose catalytic mechanisms for GenB3 and GenB4, which shed new light on the already unrivalled catalytic versatility of PLP-dependent enzymes.

microbiology↗

bioRxiv2020

Chemical validation of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase using fragment linking and CRISPR interference

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify the first inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a KD < 20 M and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biochemistry↗

bioRxiv2020

Using a fragment-based approach to identify novel chemical scaffolds targeting the dihydrofolate reductase (DHFR) from Mycobacterium tuberculosis

Dihydrofolate reductase (DHFR), a key enzyme involved in folate metabolism, is a widely explored target in the treatment of cancer, immune diseases, bacteria and protozoa infections. Although several antifolates have proved successful in the treatment of infectious diseases, none have been developed to combat tuberculosis, despite the essentiality of M. tuberculosis DHFR (MtDHFR). Herein, we describe an integrated fragment-based drug discovery approach to target MtDHFR that has identified hits with scaffolds not yet explored in any previous drug design campaign for this enzyme. The application of a SAR by catalog strategy of an in house library for one of the identified fragments has led to a series of molecules that bind MtDHFR with low micromolar affinities. Crystal structures of MtDHFR in complex with compounds of this series demonstrated a novel binding mode that differs from other DHFR antifolates, thus opening perspectives for the development of novel and relevant MtDHFR inhibitors.

biochemistry↗

bioRxiv2020

Trypanosomatid selenophosphate synthetase structure, function and interaction with selenocysteine lyase

Early branching eukaryotes have been used as models to study the evolution of cellular molecular processes. Strikingly, human parasite of the Trypanosomatidae family (T. brucei, T. cruzi and L. major) conserve the complex machinery responsible for selenocysteine biosynthesis and incorporation in selenoproteins (SELENOK/SelK, SELENOT/SelT and SELENOTryp/SelTryp), although these proteins do not seem to be essential for parasite viability under laboratory controlled conditions. Selenophosphate synthetase (SEPHS/SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the formation of selenophosphate, the biological selenium donor for selenocysteine synthesis. We solved the crystal structure of the L. major selenophosphate synthetase and confirmed that its dimeric organization is functionally important throughout the domains of life. We also demonstrated its interaction with selenocysteine lyase (SCLY) and showed that it is not present in other stable complexes involved in the selenocysteine pathway, namely the phosphoseryl-tRNASec kinase (PSTK)-Sec-tRNASec synthase (SEPSECS) and the tRNASec-specific elongation factor (eEFSec)-ribosome. Endoplasmic reticulum stress with ditiothreitol (DTT) or tunicamycin upon selenophosphate synthetase ablation in procyclic T. brucei cells led to a growth defect. On the other hand, only DTT presented a negative effect in bloodstream T. brucei expressing selenophosphate synthetase-RNAi. Although selenoprotein T (SELENOT) was dispensable for both forms of the parasite, SELENOT-RNAi procyclic T. brucei cells were sensitive to DTT. Together, our data suggest a role for the T. brucei selenophosphate synthetase in regulation of the parasites ER stress response. SynopsisSelenium is both a toxic compound and a micronutrient. As a micronutrient, it participates in the synthesis of specific proteins, selenoproteins, as the amino acid selenocysteine. The synthesis of selenocysteine is present in organisms ranging from bacteria to humans. The protozoa parasites of the Trypanosomatidae family, that cause major tropical diseases, conserve the complex machinery responsible for selenocysteine biosynthesis and incorporation in selenoproteins. However, this pathway has been considered dispensable for the protozoa cells. This has intrigued us, and lead to question that if maintained in the cell it should be under selective pressure and therefore be necessary. Also, since the intermediate products of selenocysteine synthesis are toxic to the cell, it has been proposed that these compounds need to be sequestered from the cytoplasm. Therefore, extensive and dynamic protein-protein interactions must happen to deliver those intermediates along the pathway. In this study we have investigated the molecular and structural interactions of different proteins involved in selenocystein synthesis and describe its involvement in the endoplasmic reticulum protection to oxidative stress. Our results also show how the interaction of different proteins leads to the protection of the cell against the toxic effects of seleium compounds during selenocysteine synthesis.

molecular biology↗

bioRxiv2019

Mycobacterial OtsA structures reveal an allosteric site regulated by 2-oxoglutarate and 2-phosphoglycerate and the mechanism for substrate preference.

Trehalose is an essential disaccharide for mycobacteria and a key constituent of several cell wall glycolipids with fundamental roles in pathogenesis. Mycobacteria possess two pathways for trehalose biosynthesis. However, only the OtsAB pathway was found to be essential in M. tuberculosis, with marked growth and virulence defects of OtsA mutants and strict essentiality of OtsB2. Herein, we report the first mycobacterial OtsA structures from M. thermoresistibile in both apo and ligand-bound forms. Structural information reveals three key residues in the mechanism of substrate preference that were further confirmed by site-directed mutagenesis. Additionally, we identify 2-oxoglutarate and 2-phosphoglycerate as allosteric regulators of OtsA. The structural analysis in this work strongly contributed to define the mechanisms for feedback inhibition, show different conformational states of the enzyme and map a new allosteric site.

biochemistry↗