Gating interactions steer loop conformational changes in the active site of the L1 metallo-β-lactamase

β-Lactam antibiotics are the most important and widely used antibacterial agents across the world. However, the widespread dissemination of β-lactamases among pathogenic bacteria limits the efficacy of β-lactam antibiotics. This has created a major public health crisis. The use of β-lactamase inhibitors has proven useful in restoring the activity of β-lactam antibiotics, yet, effective clinically approved inhibitors against class B metallo-β-lactamases are not available. L1, a class B3 enzyme expressed by Stenotrophomonas maltophilia, is a significant contributor to the β-lactam resistance displayed by this opportunistic pathogen. Structurally, L1 is a tetramer with two elongated loops, α3-β7 and β12-α5, present around the active site of each monomer. Residues in these two loops influence substrate/inhibitor binding. To study how the conformational changes of the elongated loops affect the active site in each monomer, enhanced sampling molecular dynamics simulations were performed, Markov State Models were built, and convolutional variational autoencoder-based deep learning was applied. The key identified residues (D150a, H151, P225, Y227, and R236) were mutated and the activity of the generated L1 variants was evaluated in cell-based experiments. The results demonstrate that there are extremely significant gating interactions between α3-β7 and β12-α5 loops. Taken together, the gating interactions with the conformational changes of the key residues play an important role in the structural remodeling of the active site. These observations offer insights into the potential for novel drug development exploiting these gating interactions

Dorfman-Chanarin syndrome: A rare neutral lipid storage disease

Dorfman-Chanarin syndrome is a rare neutral lipid storage disorder characterized by ichthyosis, lipid vacuolations in peripheral leucocytes, and multisystem involvement. It is an autosomal recessive disorder caused by mutations in the CGI-58 gene. A total of 42 cases have been reported worldwide till February 2009 out of which 4 have been previously reported from India. We report a case of a 20-month-old male with congenital ichthyosis, organomegaly, and bilateral cryptorchidism. Examination of the peripheral smear revealed lipid vacuoles in the leucocytes consistent with Jordan′s anomaly, which was confirmed by transmission electron microscopy. Liver biopsy revealed micronodular cirrhosis with macrovesicular steatosis while skin biopsy showed ichthyosis vulgaris. Dorfman-Chanarin syndrome was diagnosed on the basis of clinical and laboratory criteria with certain unreported manifestations. Dietary modifications were instituted and followed up after 1 year with promising results. This emphasizes the importance of neonatal screening for lipid vacuolations in peripheral blood in all cases of congenital ichthyosis

Revisiting the Mechanism of the Triosephosphate Isomerase Reaction: The Role of the Fully Conserved Glutamic Acid 97 Residue

An analysis of 503 available triosephosphate isomerase sequences revealed nine fully conserved residues. Of these, four residues-K12, H95, E97 and E165-are capable of proton transfer and are all arrayed around the dihydroxyacetone phosphate substrate in the three-dimensional structure. Specific roles have been assigned to the residues K12, H95 and E165, but the nature of the involvement of E97 has not been established. Kinetic and structural characterization is reported for the E97Q and E97D mutants of Plasmodium falciparum triosephosphate isomerase (Pf TIM). A 4000-fold reduction in k(cat) is observed for E97Q, whereas the E97D mutant shows a 100-fold reduction. The control mutant, E165A, which lacks the key catalytic base, shows an approximately 9000-fold drop in activity. The integrity of the overall fold and stability of the dimeric structure have been demonstrated by biophysical studies. Crystal structures of E97Q and E97D mutants have been determined at 2.0 angstrom resolution. In the case of the isosteric replacement of glutamic acid by glutamine in the E97Q mutant a large conformational change for the critical K12 side chain is observed, corresponding to a trans-to-gauche transition about the C gamma-C delta (chi(3)) bond. In the E97D mutant, the K12 side chain maintains the wild-type orientation, but the hydrogen bond between K12 and D97 is lost. The results are interpreted as a direct role for E97 in the catalytic proton transfer cycle. The proposed mechanism eliminates the need to invoke the formation of the energetically unfavourable imidazolate anion at H95, a key feature of the classical mechanism

Mortality indicators with clinical profile of multisystem inflammatory syndrome in children during SARS-CoV-2 second wave in India: A tertiary referral center experience

Background: Post-COVID-19 cases are being reported with features of hyperinflammatory state causing multiple system dysfunctions in previously healthy children. Objectives: To describe clinical characteristics, laboratory, and radiological profile of children affected with COVID-19-related multisystem inflammatory syndrome postsecond wave in India and compare them with respect to adverse outcome. Materials and Methods: This prospective, observational study was conducted in the department of pediatrics of a tertiary care center in Eastern India over a period of 3 months. Demographic data, clinical details, biochemical parameters, and treatment with clinical outcome were recorded. Children who survived the clinical course were compared with those died during hospital stay. Results: Thirty-five children with a median age of 4.8 (3.9) years were included who were admitted between June 16 and September 15, 2021. Only 17.14% had reverse transcription-polymerase chain reaction positivity previously with 77.14% had positive COVID-19 serology. Most common features were fever (100%), edema (68.6%), gastrointestinal (71.4%), mucocuteneous (65.7%), cardiovascular (57.1%), and neurological symptoms (45.7%). Twenty (57.1%) children had shock at presentation. Decreased ejection fraction (<55%) was the most common echocardiographic feature (37.14%) followed by coronary dilatation (20%). Majority (77.14%) of the patients required intensive care with inotrope requirement in 62.86% cases. Forty percent patients were intubated with mean duration of 9.94 (±10.5) days. All patients received methylprednisolone and 76% were given intravenous immunoglobulin. Tocilizumab was used in three patients. Nine patients died (25.7%) with overall median pediatric intensive care unit stay of 13 (14) days. Conclusion: Of the parameters described, we have found shock, heart failure, neurological involvement at presentation, infancy, and laboratory parameters such as C-reactive protein, CPK, D-Dimer, and lactate dehydrogenase were the predictors of mortality

Probing the role of the fully conserved Cys126 in triosephosphate isomerase by site-specific mutagenesis - distal effects on dimer stability

DatabaseStructural data are available in the Protein Data Bank under the accession numbers <externallink id=''http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3PVF '' type=''url''>3PVF, <externallink id=''http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3PY2 '' type=''url''>3PY2, and <externallink id=''http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3PWA '' type=''url''>3PWA. Structured digital abstract <externallink id=''http://www.uniprot.org/uniprot/Q07412'' type=''url''>Tim <externallink id=''http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407'' type=''url''>binds to <externallink id=''http://www.uniprot.org/uniprot/Q07412'' type=''url''>Tim by <externallink id=''http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114'' type=''url''>x-ray crystallography <externallink id=''http://mint.bio.uniroma2.it/mint/search/interaction.do?interactio nAc=MINT-8149884'' type=''url''>(View interaction)

Probing the role of the fully conserved Cys126 in triosephosphate isomerase by site-specific mutagenesis - distal effects on dimer stability

Cys126 is a completely conserved residue in triosephosphate isomerase that is proximal to the active site but has been ascribed no specific role in catalysis. A previous study of the C126S and C126A mutants of yeast TIM reported substantial catalytic activity for the mutant enzymes, leading to the suggestion that this residue is implicated in folding and stability [Gonzalez-Mondragon E et al. (2004) Biochemistry43, 3255-3263]. We re-examined the role of Cys126 with the Plasmodium falciparum enzyme as a model. Five mutants, C126S, C126A, C126V, C126M, and C126T, were characterized. Crystal structures of the 3-phosphoglycolate-bound C126S mutant and the unliganded forms of the C126S and C126A mutants were determined at a resolution of 1.7-2.1 Å. Kinetic studies revealed an approximately five-fold drop in k<SUB>cat</SUB> for the C126S and C126A mutants, whereas an approximately 10-fold drop was observed for the other three mutants. At ambient temperature, the wild-type enzyme and all five mutants showed no concentration dependence of activity. At higher temperatures (&gt; 40 ° C), the mutants showed a significant concentration dependence, with a dramatic loss in activity below 15 μ m. The mutants also had diminished thermal stability at low concentration, as monitored by far-UV CD. These results suggest that Cys126 contributes to the stability of the dimer interface through a network of interactions involving His95, Glu97, and Arg98, which form direct contacts across the dimer interface