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Protein Engineering, Design & Selection: PEDS

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https://www.readbyqxmd.com/read/28100651/stabilization-of-bacillus-circulans-xylanase-by-combinatorial-insertional-fusion-to-a-thermophilic-host-protein
#1
Vandan Shah, Brennal Pierre, Tamari Kirtadze, Seung Shin, Jin Ryoun Kim
High thermostability of an enzyme is critical for its industrial application. While many engineering approaches such as mutagenesis have enhanced enzyme thermostability, they often suffer from reduced enzymatic activity. A thermally stabilized enzyme with unchanged amino acids is preferable for subsequent functional evolution necessary to address other important industrial needs. In the research presented here, we applied insertional fusion to a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) in order to improve the thermal stability of Bacillus circulans xylanase (BCX)...
January 17, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28073960/ligand-characterization-of-cyp4b1-isoforms-modified-for-high-level-expression-in-escherichia-coli-and-hepg2-cells
#2
Katharina Roellecke, Vera D Jäger, Veselin H Gyurov, John P Kowalski, Stephanie Mielke, Allan E Rettie, Helmut Hanenberg, Constanze Wiek, Marco Girhard
Human CYP4B1, a cytochrome P450 monooxygenase predominantly expressed in the lung, inefficiently metabolizes classical CYP4B1 substrates, such as the naturally occurring furan pro-toxin 4-ipomeanol (4-IPO). Highly active animal forms of the enzyme convert 4-IPO to reactive alkylating metabolite(s) that bind(s) to cellular macromolecules. By substitution of 13 amino acids, we restored the enzymatic activity of human CYP4B1 toward 4-IPO and this modified cDNA is potentially valuable as a suicide gene for adoptive T-cell therapies...
January 10, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28062648/dual-chemistry-catalyzed-by-human-acireductone-dioxygenase
#3
Aditi R Deshpande, Thomas C Pochapsky, Gregory A Petsko, Dagmar Ringe
Acireductone dioxygenase (ARD) from the methionine salvage pathway of Klebsiella oxytoca is the only known naturally occurring metalloenzyme that catalyzes different reactions in vivo based solely on the identity of the divalent transition metal ion (Fe(2+) or Ni(2+)) bound in the active site. The iron-containing isozyme catalyzes the cleavage of substrate 1,2-dihydroxy-3-keto-5-(thiomethyl)pent-1-ene (acireductone) by O2 to formate and the ketoacid precursor of methionine, whereas the nickel-containing isozyme uses the same substrates to catalyze an off-pathway shunt to form methylthiopropionate, carbon monoxide and formate...
January 5, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28062647/on-the-effect-of-alkaline-ph-and-cofactor-availability-in-the-conformational-and-oligomeric-state-of-escherichia-coli-glutamate-decarboxylase
#4
F Giovannercole, C Mérigoux, C Zamparelli, D Verzili, G Grassini, M Buckle, P Vachette, D De Biase
Escherichia coli glutamate decarboxylase (EcGad) is a homohexameric pyridoxal 5'-phosphate (PLP)-dependent enzyme. It is the structural component of the major acid resistance system that protects E. coli from strong acid stress (pH < 3), typically encountered in the mammalian gastrointestinal tract. In fact EcGad consumes one proton/catalytic cycle while yielding γ-aminobutyrate and carbon dioxide from the decarboxylation of l-glutamate. Two isoforms of Gad occur in E. coli (GadA and GadB) that are 99% identical in sequence...
January 5, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28062646/generation-of-human-bispecific-common-light-chain-antibodies-by-combining-animal-immunization-and-yeast-display
#5
Simon Krah, Christian Schröter, Carla Eller, Laura Rhiel, Nicolas Rasche, Jan Beck, Carolin Sellmann, Ralf Günther, Lars Toleikis, Björn Hock, Harald Kolmar, Stefan Becker
Bispecific antibodies (bsAbs) pave the way for novel therapeutic modes of action along with potential benefits in several clinical applications. However, their generation remains challenging due to the necessity of correct pairings of two different heavy and light chains and related manufacturability issues. We describe a generic approach for the generation of fully human IgG-like bsAbs. For this, heavy chain repertoires from immunized transgenic rats were combined with either a randomly chosen common light chain or a light chain of an existing therapeutic antibody and screened for binders against tumor-related targets CEACAM5 and CEACAM6 by yeast surface display...
January 5, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28062645/crystallographic-substrate-binding-studies-of-leishmania-mexicana-scp2-thiolase-type-2-unique-features-of-oxyanion-hole-1
#6
Rajesh K Harijan, Tiila-Riikka Kiema, Shahan M Syed, Imran Qadir, Muriel Mazet, Frédéric Bringaud, Paul A M Michels, Rik K Wierenga
Structures of the C123A variant of the dimeric Leishmania mexicana SCP2-thiolase (type-2) (Lm-thiolase), complexed with acetyl-CoA and acetoacetyl-CoA, respectively, are reported. The catalytic site of thiolase contains two oxyanion holes, OAH1 and OAH2, which are important for catalysis. The two structures reveal for the first time the hydrogen bond interactions of the CoA-thioester oxygen atom of the substrate with the hydrogen bond donors of OAH1 of a CHH-thiolase. The amino acid sequence fingerprints ( C: xS, N: EAF, G H: P) of three catalytic loops identify the active site geometry of the well-studied CNH-thiolases, whereas SCP2-thiolases (type-1, type-2) are classified as CHH-thiolases, having as corresponding fingerprints C: xS, H: DCF and G H: P...
January 5, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28062644/ligand-induced-conformational-changes-in-prolyl-oligopeptidase-a-kinetic-approach
#7
R Van Elzen, E Schoenmakers, I Brandt, P Van Der Veken, A M Lambeir
Most kinetic studies of prolyl oligopeptidase (PREP) were performed with the porcine enzyme using modified peptide substrates. Yet recent biophysical studies used the human homolog. Therefore, the aim of this study was to compare the kinetic behavior of human and porcine PREP, as well as to find a suitable method to study enzyme kinetics with an unmodified biological substrate. It was found that human PREP behaves identically to the porcine homolog, displaying a double bell-shaped pH profile and a pH-dependent solvent kinetic isotope effect of the kcat/Km, features that set it apart from the related exopeptidase dipeptidyl peptidase IV (DPP IV)...
January 5, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28044007/modification-of-the-peroxygenative-peroxidative-activity-ratio-in-the-unspecific-peroxygenase-from-agrocybe-aegerita-by-structure-guided-evolution
#8
Diana M Mate, Miguel A Palomino, Patricia Molina-Espeja, Javier Martin-Diaz, Miguel Alcalde
Unspecific peroxygenase (UPO) is a heme-thiolate peroxidase capable of performing with high-selectivity C-H oxyfunctionalizations of great interest in organic synthesis through its peroxygenative activity. However, the convergence of such activity with an unwanted peroxidative activity encumbers practical applications. In this study, we have modified the peroxygenative:peroxidative activity ratio (P:p ratio) of UPO from Agrocybe aegerita by structure-guided evolution. Several flexible loops (Glu1-Pro35, Gly103-Asp131, Ser226-Gly243, Gln254-Thr276 and Ty293-Arg327) were selected on the basis on their B-factors and ΔΔG values...
January 1, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28035012/deep-sequencing-of-phage-displayed-peptide-libraries-reveals-sequence-motif-that-detects-norovirus
#9
Amy M Hurwitz, Wanzhi Huang, Mary K Estes, Robert L Atmar, Timothy Palzkill
Norovirus infections are the leading cause of non-bacterial gastroenteritis and result in about 21 million new cases and $2 billion in costs per year in the United States. Existing diagnostics have limited feasibility for point-of-care applications, so there is a clear need for more reliable, rapid, and simple-to-use diagnostic tools in order to contain outbreaks and prevent inappropriate treatments. In this study, a combination of phage display technology, deep sequencing and computational analysis was used to identify 12-mer peptides with specific binding to norovirus genotype GI...
December 28, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28007937/directed-evolution-of-p450cin-for-mediated-electron-transfer
#10
Ketaki D Belsare, Thomas Horn, Anna Joëlle Ruff, Ronny Martinez, Anders Magnusson, Dirk Holtmann, Jens Schrader, Ulrich Schwaneberg
Directed evolution is a powerful method to optimize enzyme properties for application demands. Interesting targets are P450 monooxygenases which catalyze the stereo- and regiospecific hydroxylation of chemically inert C-H bonds. Synthesis employing P450s under cell-free reaction conditions is limited by low total turnover numbers, enzyme instability, low product yields and the requirement of the expensive co-factor NADPH. Bioelectrocatalysis is an alternative to replace NADPH in cell-free P450-catalyzed reactions...
December 22, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27999093/a-robust-cosolvent-compatible-halohydrin-dehalogenase-by-computational-library-design
#11
Hesam Arabnejad, Marco Dal Lago, Peter A Jekel, Robert J Floor, Andy-Mark W H Thunnissen, Anke C Terwisscha van Scheltinga, Hein J Wijma, Dick B Janssen
To improve the applicability of halohydrin dehalogenase as a catalyst for reactions in the presence of organic cosolvents, we explored a computational library design strategy (Framework for Rapid Enzyme Stabilization by Computational libraries) that involves discovery and in silico evaluation of stabilizing mutations. Energy calculations, disulfide bond predictions and molecular dynamics simulations identified 218 point mutations and 35 disulfide bonds with predicted stabilizing effects. Experiments confirmed 29 stabilizing point mutations, most of which were located in two distinct regions, whereas introduction of disulfide bonds was not effective...
December 19, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27999092/a-theoretical-study-on-the-reactivity-of-the-mo-cu-containing-carbon-monoxide-dehydrogenase-with-dihydrogen
#12
Raffaella Breglia, Maurizio Bruschi, Ugo Cosentino, Luca De Gioia, Claudio Greco, Toshiko Miyake, Giorgio Moro
The Mo/Cu-dependent CO dehydrogenase from Oligotropha carboxidovorans is an enzyme that is able to catalyze CO oxidation to CO2; moreover, it can also oxidize H2, thus eliciting a characteristic EPR signal. Interestingly, the Ag-substituted enzyme form proved unable to catalyze H2 oxidation. In the present contribution, we characterized the reactivity of the enzyme with H2 by quantum-chemical calculations. It was found that dihydrogen binding to the wild-type enzyme requires significant structural rearrangements of the active site Theoretical EPR spectra for plausible H2-bound models of the partially reduced, paramagnetic active site are also presented and compared with the experimental counterpart...
December 19, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27986921/development-of-a-cancer-marker-activated-enzymatic-switch-from-the-herpes-simplex-virus-thymidine-kinase
#13
Nirav Y Shelat, Sidhartha Parhi, Marc Ostermeier
Discovery of new cancer biomarkers and advances in targeted gene delivery mechanisms have made gene-directed enzyme prodrug therapy (GDEPT) an attractive method for treating cancer. Recent focus has been placed on increasing target specificity of gene delivery systems and reducing toxicity in non-cancer cells in order to make GDEPT viable. To help address this challenge, we have developed an enzymatic switch that confers higher prodrug toxicity in the presence of a cancer marker. The enzymatic switch was derived from the herpes simplex virus thymidine kinase (HSV-TK) fused to the CH1 domain of the p300 protein...
December 15, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27986920/annexin-directed-%C3%AE-glucuronidase-for-the-targeted-treatment-of-solid-tumors
#14
Katrin P Guillen, Eliza A Ruben, Needa Virani, Roger G Harrison
Enzyme prodrug therapy has the potential to remedy the lack of selectivity associated with the systemic administration of chemotherapy. However, most current systems are immunogenic and constrained to a monotherapeutic approach. We developed a new class of fusion proteins centered about the human enzyme β-glucuronidase (βG), capable of converting several innocuous prodrugs into chemotherapeutics. We targeted βG to phosphatidylserine on tumor cells, tumor vasculature and metastases via annexin A1/A5. Phosphatidylserine shows promise as a universal marker for solid tumors and allows for tumor type-independent targeting...
December 15, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27980121/chimeric-approach-for-narrowing-a-membrane-inserting-region-within-human-perforin
#15
Amy E Neely, Kimberly A Mandigo, Rebekah L Robinson, Traci L Ness, Mitch H Weiland
Perforin is a pore-forming, immune protein that functions to deliver an apoptotic cocktail of proteins into a target pathogen. Recent studies of the bacterial cholesterol-dependent cytolysins (CDCs) have provided a model for perforin's pore-forming mechanism. Both perforin and CDC family members share a conserved β-sheet flanked by two clusters of α-helices. Within the CDCs, these helices refold into two transmembrane β-hairpins, TMH1 and TMH2. Based upon structural conservation and electron microscopy imaging, the analogous helices within perforin are predicted to also be membrane inserting; however, these regions are approximately twice the length of the CDC TMHs...
December 15, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27980120/production-and-characterization-of-functional-recombinant-hybrid-heteropolymers-of-camel-hepcidin-and-human-ferritin-h-and-l-chains
#16
Mohamed Boumaiza, Fernando Carmona, Maura Poli, Michela Asperti, Alessandra Gianoncelli, Michela Bertuzzi, Paola Ruzzenenti, Paolo Arosio, Mohamed Nejib Marzouki
Hepcidin is a liver-synthesized hormone that plays a central role in the regulation of systemic iron homeostasis. To produce a new tool for its functional properties the cDNA coding for camel hepcidin-25 was cloned at the 5'end of human FTH sequence into the pASK-IBA43plus vector for expression in Escherichiacoli The recombinant fusion hepcidin-ferritin-H subunit was isolated as an insoluble iron-containing protein. When alone it did not refold in a 24-mer ferritin molecule, but it did when renatured together with H- or L-ferritin chains...
December 15, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27903763/enzyme-activation-through-the-utilization-of-intrinsic-dianion-binding-energy
#17
REVIEW
T L Amyes, M M Malabanan, X Zhai, A C Reyes, J P Richard
We consider 'the proposition that the intrinsic binding energy that results from the noncovalent interaction of a specific substrate with the active site of the enzyme is considerably larger than is generally believed. An important part of this binding energy may be utilized to provide the driving force for catalysis, so that the observed binding energy represents only what is left over after this utilization' [Jencks,W.P. (1975) Adv. Enzymol. Relat. Areas. Mol. Biol., 43: , 219-410]. The large ~12 kcal/mol intrinsic substrate phosphodianion binding energy for reactions catalyzed by triosephosphate isomerase (TIM), orotidine 5'-monophosphate decarboxylase and glycerol-3-phosphate dehydrogenase is divided into 4-6 kcal/mol binding energy that is expressed on the formation of the Michaelis complex in anchoring substrates to the respective enzyme, and 6-8 kcal/mol binding energy that is specifically expressed at the transition state in activating the respective enzymes for catalysis...
November 29, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27986919/generation-of-inhibitory-monoclonal-antibodies-targeting-matrix-metalloproteinase-14-by-motif-grafting-and-cdr-optimization
#18
Dong Hyun Nam, Kuili Fang, Carlos Rodriguez, Tyler Lopez, Xin Ge
Matrix metalloproteinase-14 (MMP-14) plays important roles in cancer metastasis, and the failures of broad-spectrum MMP compound inhibitors in clinical trials suggested selectivity is critical. By grafting an MMP-14 specific inhibition motif into complementarity determining region (CDR)-H3 of antibody scaffolds and optimizing other CDRs and the sequences that flank CDR-H3, we isolated a Fab 1F8 showing a binding affinity of 8.3 nM with >1000-fold enhancement on inhibition potency compared to the peptide inhibitor...
February 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27899437/allosteric-control-of-antibody-prion-recognition-through-oxidation-of-a-disulfide-bond-between-the-ch-and-cl-chains
#19
Jun Zhao, Ruth Nussinov, Buyong Ma
Molecular details of the recognition of disordered antigens by their cognate antibodies have not been studied as extensively as folded protein antigens and much is still unknown. To follow the conformational changes in the antibody and cross-talk between its subunits and with antigens, we performed molecular dynamics (MD) simulations of the complex of Fab and prion-associated peptide in the apo and bound forms. We observed that the inter-chain disulfide bond in constant domains restrains the conformational changes of Fab, especially the loops in the CH1 domain, resulting in inhibition of the cross-talk between Fab subdomains that thereby may prevent prion peptide binding...
January 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27887027/expansion-of-the-substrate-range-of-the-gentisate-1-2-dioxygenase-from-corynebacterium-glutamicum-for-the-conversion-of-monohydroxylated-benzoates
#20
Erik Eppinger, Andreas Stolz
The gentisate 1,2-dioxygenases (GDOs) from Corynebacterium glutamicum and various other organisms oxidatively cleave the aromatic nucleus of gentisate (2,5-dihydroxybenzoate), but are not able to convert salicylate (2-hydroxybenzoate). In contrast, the α-proteobacterium Pseudaminobacter salicylatoxidans synthesises an enzyme ('salicylate dioxygenase', SDO) which cleaves gentisate, but also (substituted) salicylate(s). Sequence comparisons showed that the SDO belongs to a group of GDOs mainly originating from Gram-positive bacteria which also include the GDO from C...
January 2017: Protein Engineering, Design & Selection: PEDS
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