Lantibiotics represent a large untapped pipeline of attractive scaffolds for the development of novel antibiotics. Saturation mutagenesis was employed to substitute every amino acid of a lantibiotic ...called mutacin 1140 (MU1140), creating an unbiased expression library of 418 variants that was used to study the permissiveness to mutagenesis and the “drugability” of several compounds. Contrasting previous reports, the results from this study supported that not all residues involved in lanthionine bridge formation were critical for maintaining optimal activity. While substitutions in lanthionine bridges in Ring A, C, and D invariably lead to inactive variants, permissive substitutions in Abu8 and Ala11 (Ring B) were observed, albeit infrequently. Further, the data generated suggested that the unsaturated bond from Dha5 (Ser5) may not be critically involved in Lipid‐II binding but still important for conferring optimal activity. This study identified additional permissive mutations of Ser5, including Ser5His, Ser5Met, Ser5Gln, and Ser5Leu. In contrast, no permissive substitutions were identified for Dhb14, which suggested that this residue may be critical for optimal activity. Novel blueprints are proposed for directing further development of MU1140 variants and other lantibiotics, which may enable the rational design, development, manufacture, and formulation of an entirely new class of anti‐infectives.
Lantibiotics represent a large untapped pipeline of attractive scaffolds for the development of novel antibiotics. Saturation mutagenesis was employed to substitute every amino acid of the core peptide of a lantibiotic called mutacin 1140, creating an unbiased expression library of 418 variants that was used to study the structure–activity relationship of key residues. Blueprints are proposed for directing further development of therapeutic lantibiotics, which enable the rational design, development, manufacture, and formulation of an entirely new class of anti‐infectives.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
We report the construction of a synthetic flavo-heme protein that incorporates two major physiological activities of flavoproteins: light activation of flavin analogous to DNA photolyase and rapid ...intramolecular electron transfer between the flavin and heme cofactors as in several oxidoreductases. The functional tetra-α -helix protein comprises two 62-aa helix-loop-helix subunits. Each subunit contains a single cysteine to which flavin (7-acetyl-10-methylisoalloxazine) is covalently attached and two histidines appropriately positioned for bis-his coordination of heme cofactors. Both flavins and hemes are situated within the hydrophobic core of the protein. Intramolecular electron transfer from flavosemiquinone generated by photoreduction from a sacrificial electron donor in solution was examined between protoporphyrin IX and 1-methyl-2-oxomesoheme XIII. Laser pulse-activated electron transfer from flavin to meso heme occurs on a 100-ns time scale, with a favorable free energy of approximately -100 meV. Electron transfer from flavin to the lower potential protoporphyrin IX, with an unfavorable free energy, can be induced after a lag phase under continuous light illumination. Thus, the supporting peptide matrix provides an excellent framework for the positioning of closely juxtaposed redox groups capable of facilitating intramolecular electron transfer and begins to clarify in a simplified and malleable system the natural engineering of flavoproteins.
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Small molecule screening, the systematic encounter of biology space with chemical space, has provoked the emergence of a whole industry that recreates itself by constant iterative improvements to ...this process. The authors describe an approach to tackle the problem for one of the most time-consuming steps in the execution of a screening campaign, namely, the reformatting of high-throughput screening test compounds from master plates to daughter assay plates used in the execution of the screen. Through an engineered storage procedure, they prepare plates ahead of the screening process with the respective compounds in a ready-to-use format. They show the biological inertness of the method and how it facilitates efficient recovery of compound activity. This uncoupling of normally interconnected processes provides time and compound savings, avoids repeated freeze-thaw cycles of compound solutions, and removes the problems associated with the DMSO sensitivity of certain assays types. (Journal of Biomolecular Screening 2005:573-580)
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The effects of various mechanisms of metalloporphyrin reduction potential modulation were investigated experimentally using a robust, well-characterized heme protein maquette, synthetic protein ...scaffold H10A24 {CH3 CONH-CGGGELWKL·HEELLKK·FEELLKL·AEERLKK·L-CONH2 }22. Removal of the iron porphyrin macrocycle from the high dielectric aqueous environment and sequestration within the hydrophobic core of the H10A24 maquette raises the equilibrium reduction midpoint potential by 36−138 mV depending on the hydrophobicity of the metalloporphyrin structure. By incorporating various natural and synthetic metalloporphyrins into a single protein scaffold, we demonstrate a 300-mV range in reduction potential modulation due to the electron-donating/withdrawing character of the peripheral macrocycle substituents. Solution pH is used to modulate the metalloporphyrin reduction potential by 160 mV, regardless of the macrocycle architecture, by controlling the protonation state of the glutamate involved in partial charge compensation of the ferric heme. Attempts to control the reduction potential by inserting charged amino acids into the hydrophobic core at close proximity to the metalloporphyrin lead to varied success, with H10A24-L13E lowering the E m8.5 by 40 mV, H10A24-E11Q raising it by 50 mV, and H10A24-L13R remaining surprisingly unaltered. Modifying the charge of the adjacent metalloporphyrin, +1 for iron(III) protoporphyrin IX or neutral for zinc(II) protoporphyrin IX resulted in a loss of 70 mV Fe(III)PPIX+ − Fe(III)PPIX+ interaction observed in maquettes. Using these factors in combination, we illustrate a 435-mV variation of the metalloporphyrin reduction midpoint potential in a simple heme maquette relative to the about 800-mV range observed for natural cytochromes. Comparison between the reduction potentials of the heme maquettes and other de novo designed heme proteins reveals global trends in the E m values of synthetic cytochromes.
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IJS, KILJ, NUK, PNG, UL, UM
The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase, EC 1.1.2.3) are connected by a typical hinge peptide. To probe the importance of the structural integrity of the ...hinge region for efficient intraprotein electron transfer, three mutant enzymes have been constructed: H delta 3 Sharp, R. E., White, P., Chapman, S. K., & Reid, G. A. (1994) Biochemistry 33, 5115-5120, H delta 6, and H delta 9 in which three, six, and nine amino acids, respectively, were deleted from the hinge region. Intraprotein electron transfer was investigated by steady-state and stopped-flow kinetic analyses. All three hinge-deletion enzymes remained good L-lactate dehydrogenases, as was evident from steady-state experiments with ferricyanide as the electron acceptor and from stopped-flow experiments monitoring flavin reduction. The global effect of these deletions is to lower the enzyme's effectiveness as a cytochrome c reductase. This property of H delta 6 and H delta 9 flavocytochromes b2 is manifested at the first interdomain electron-transfer step (fully reduced FMN-->heme electron transfer), where the rate of heme reduction is the same within experimental error as the steady-state rate of cytochrome c reduction. Thus, interdomain electron transfer is rate limiting in the case of these two hinge-deletion enzymes compared to the wild-type enzyme, where alpha H abstraction from C-2 of L-lactate still contributes substantially to rate limitation. The situation for H delta 3 is more complicated, with more than one interdomain electron-transfer step being affected. Kinetic data, along with the measured deuterium kinetic isotope effects, are discussed in the context of the flavocytochrome b2 catalytic cycle and show that complete structural integrity within the hinge region is essential for efficient interdomain communication.
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IJS, KILJ, NUK, PNG, UL, UM
Protein-mediated electron transfer is a key process in nature. Many of the proteins involved in such electron transfers are complex and contain a number of redox-active cofactors. The very complexity ...of these multi-centre redox proteins has made it difficult to fully understand the various electron transfer events they catalyse. This is sometimes because the electron transfer steps themselves are gated or coupled to other processes such as proton transfer. However, with the molecular structures of many of these proteins now available it is possible to probe these electron transfer reactions at the molecular level. It is becoming apparent that many of these multi-centre redox proteins have rather subtle and elegant ways for regulating electron transfer. The purpose of this article is to illustrate how nature has used different approaches to control electron transfer in a number of different systems. Illustrative examples include: thermodynamic control of electron transfer in flavocytochromes
b
2 and P450 BM3; a novel control mechanism involving calmodulin-binding-dependent electron transfer in neuronal nitric oxide synthase; the probable gating of electron transfer by ATP hydrolysis in nitrogenase; conformational gating of electron transfer in cytochrome
cd
1; the regulation of electron transfer by protein dynamics in the cytochrome
bc
1 complex; and finally the coupling of electron transfer to proton transfer in cytochrome
c oxidase.
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IJS, IMTLJ, KILJ, KISLJ, NUK, SAZU, SBCE, SBJE, UL, UM, UPUK
We report the construction of a synthetic flavo-heme protein that incorporates two major physiological activities of flavoproteins: light activation of flavin analogous to DNA photolyase and rapid ...intramolecular electron transfer between the flavin and heme cofactors as in several oxidoreductases. The functional tetra-alpha-helix protein comprises two 62-aa helix-loop-helix subunits.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
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