03098nas a2200289   4500008004100000022001400041245018200055210006900237260006700306300001400373490000700387520197200394653002802366653002902394653002702423653002402450653002402474653001802498100001802516700002702534700002602561700002602587700002502613700002702638700001902665856012402684       2015                        eng d  a0961-836800aStructure mediation in substrate binding and post-translational processing of penicillin acylases: Information from mutant structures of Kluyvera citrophila penicillin G acylase0 aStructure mediation in substrate binding and posttranslational p  a111 RIVER ST, HOBOKEN 07030-5774, NJ USAbWILEY-BLACKWELLcOCT  a1660-16700 v243 a<p>Penicillin acylases are industrially important enzymes for the production of 6-APA, which is used extensively in the synthesis of secondary antibiotics. The enzyme translates into an inactive single chain precursor that subsequently gets processed by the removal of a spacer peptide connecting the chains of the mature active heterodimer. We have cloned the penicillin G acylase from Kluyvera citrophila (KcPGA) and prepared two mutants by site-directed mutagenesis. Replacement of N-terminal serine of the beta-subunit with cysteine (Ser beta 1Cys) resulted in a fully processed but inactive enzyme. The second mutant in which this serine is replaced by glycine (Ser beta 1Gly) remained in the unprocessed and inactive form. The crystals of both mutants belonged to space group P1 with four molecules in the asymmetric unit. The three-dimensional structures of these mutants were refined at resolutions 2.8 and 2.5 angstrom, respectively. Comparison of these structures with similar structures of Escherichia coli PGA (EcPGA) revealed various conformational changes that lead to autocatalytic processing and consequent removal of the spacer peptide. The large displacements of residues such as Arg168 and Arg477 toward the N-terminal cleavage site of the spacer peptide or the conformational changes of Arg145 and Phe146 near the active site in these structures suggested probable steps in the processing dynamics. A comparison between the structures of the processed Serb1Cys mutant and that of the processed form of EcPGA showed conformational differences in residues Arg alpha 145, Phe alpha 146, and Phe beta 24 at the substrate binding pocket. Three conformational transitions of Arg alpha 145 and Phe alpha 146 residues were seen when processed and unprocessed forms of KcPGA were compared with the substrate bound structure of EcPGA. Structure mediation in activity difference between KcPGA and EcPGA toward acyl homoserine lactone (AHL) is elucidated.</p>10aacyl homoserine lactone10aautocatalytic processing10aconformational changes10aKcPGA_Ser beta 1Cys10aKcPGA_Ser beta 1Gly10aNtn hydrolase1 aChand, Deepak1 aVarshney, NishantKumar1 aRamasamy, Sureshkumar1 aPanigrahi, Priyabrata1 aBrannigan, James, A.1 aWilkinson, Anthony, J.1 aSuresh, C., G.  uhttps://library.ncl.res.in/content/structure-mediation-substrate-binding-and-post-translational-processing-penicillin-102704nas a2200253   4500008004100000022001400041245012700055210006900182260000800251300001400259490000700273520183800280653002002118653002702138653002202165653001902187653002402206653001402230100002202244700002202266700001502288700002402303856012302327       2024                        eng d  a1948-719300aPathological mutations D169G and P112H electrostatically aggravate the amyloidogenicity of the functional domain of TDP-430 aPathological mutations D169G and P112H electrostatically aggrava  cNOV  a4267-42830 v153 a<p>
	Aggregation of TDP-43 is linked to the pathogenesis of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Notably, electrostatic point mutations such as D169G and P112H, located within the highly conserved functional tandem RNA recognition motif (RRM) domains of the TDP-43 protein (TDP-43tRRM), have been identified in diseased patients as well. In this study, we address how the electrostatic mutations alter both the native state stability and aggregation propensity of TDP-43tRRM. The mutants D169G and P112H show increased chemical stability compared to the TDP-43tRRM at physiological pH. However, at low pH, both the mutants undergo a conformational change to form amyloid-like fibrils, though with variable rates-the P112H mutant being substantially faster than the other two sequences (TDP-43tRRM and D169G mutant) showing comparable rates. Moreover, among the three sequences, only the P112H mutant undergoes a strong ionic strength-dependent aggregability trend. These observations signify the substantial contribution of the excess charge of the P112H mutant to its unique aggregation process. Complementary simulated observables with atomistic resolution assign the experimentally observed sequence-, pH-, and ionic strength-dependent aggregability pattern to the degree of thermal lability of the mutation site-containing RRM1 domain and its extent of dynamical anticorrelation with the RRM2 domain whose combination eventually dictate the extent of generation of aggregation-prone partially unfolded conformational ensembles. Our choice of a specific charge-modulated pathogenic mutation-based experiment-simulation-combination approach unravels the otherwise hidden residue-wise contribution to the individual steps of this extremely complicated multistep aggregation process.</p>
10aamyloid fibrils10aconformational changes10adisease mutations10aelectrostatics10aProtein aggregation10astability1 aPillai, Meenakshi1 aPatil, Anjali, D.1 aDas, Atanu1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/pathological-mutations-d169g-and-p112h-electrostatically-aggravate-amyloidogenicity