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R127H
site-directed mutagenesis, the mutant shows 37% reduced activity compared to the wild-type enzyme
R127K
site-directed mutagenesis, the mutant shows 91% reduced activity compared to the wild-type enzyme
A56S
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kcat is 81.3% of wild-type value
D138A
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kcat is 91.5% of wild-type value
D44G/C93S/C139S/T118A
mutant constructed to improve the overexpression and purification of the molecule as well as to obtain new crystal forms. Two cysteines are replaced to bypass misfolding problems and a charged surface residue is replaced to force different molecular packings. Mutant crystallizes in space group R3 and diffracts to 1.6 A resolution
E58Q
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kcat is 70.2% of wild-type value
F113S
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kcat is 6.8% of wild-type value
F22D
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kcat is 14.5% of wild-type value
F22S
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kcat is 47.2% of wild-type value
F22V
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kcat is 26.4% of wild-type value
F22W
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kcat is 43.8% of wild-type value
F57S
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kcat is 43.8% of wild-type value
H88A
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kcat is 12% of wild-type value
H88K
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kcat is 39.5% of wild-type value
K131N
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kcat is 9.7% of wild-type value
K131R
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kcat is 29.8% of wild-type value
K135A
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kcat is 21.9% of wild-type value
N23S
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kcat is 21.9% of wild-type value
R127H
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kcat is 69.7% of wild-type value
S142L
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kcat is 62.3% of wild-type value
T80V
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kcat is 55.1% of wild-type value
W22A
single amino acid mutation located in the active site of the endogenous RibH2. Mutant lacks enzymatic activity but its stability and structure are unaltered
L119F
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weakly binds to riboflavin
W27F
the replacement of tryptophan 27 by aliphatic amino acids substantially reduces the affinity of the enzyme for riboflavin and for the substrate, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione
W27H
the replacement of tryptophan 27 by aliphatic amino acids substantially reduces the affinity of the enzyme for riboflavin and for the substrate, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione
W27I
the replacement of tryptophan 27 by aliphatic amino acids substantially reduces the affinity of the enzyme for riboflavin and for the substrate, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione
W27S
the replacement of tryptophan 27 by aliphatic amino acids substantially reduces the affinity of the enzyme for riboflavin and for the substrate, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione
W63Y
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does not bind riboflavin
W63Y/L119F
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does not bind riboflavin
R108C
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site-directed mutagenesis
R108C
site-directed mutagenesis, substitution of the arginine residue at position 108 with cysteine, which is exposed on the exterior surface of the enzyme and can be used as a site for the attachment of small molecules. Construction of an in vivo applicable magnetic resonance positive contrast agent by conjugating Gd(III)-chelating agent complexes to lumazine synthase AaLS isolated from Aquifex aeolicus, measurement of T1 relaxation times of the Gd(III)-DOTA-AaLS, overview
W27G
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does not bind riboflavin
W27G
the replacement of tryptophan 27 by aliphatic amino acids substantially reduces the affinity of the enzyme for riboflavin and for the substrate, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione
W27Y
the replacement of tryptophan 27 by aliphatic amino acids substantially reduces the affinity of the enzyme for riboflavin and for the substrate, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione
W27Y
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whereas the indole system of W27 forms a coplanar pi-complex with riboflavin, the corresponding phenyl ring in the W27Y mutant establishes only peripheral contact with the heterocyclic ring system of the bound riboflavin
additional information
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development of lumazine synthase, isolated from hyperthermophile Aquifex aeolicus, as a modular delivery nanoplatform for the targeted delivery of diagnostic and/or therapeutic molecules depending on target cancer cells, method evaluation, overview. The enzyme is engineered for its binding and transport/ligand presentation function by mutation of rg108 to Cys, and insertion the RGD4C peptide with extra linker sequences (GGGCDCRGDCFCASGGG) to the position between residues 70 and 71, where the exterior loop is formed, allowing RGD4C peptides to adopt an active cyclic form through intramolecular disulfide bonds. In parallel, the SP94 peptide (SFSIIHTPILPL) is introduced at the C-terminal end of AaLS because the C-termini are on the exterior surface based on atomic resolution structural information and the linear form of SP94 peptide being the most active, ESI-TOF MS, UV/Vis spectrophotometric, and size-exclusion chromatographic analysis, overview. The construct binds selectively to their target cells, KB or HepG2 cells. Conjugation of bifunctional N-(3,4-dihydroxyphenethyl)-3-maleimido-propanamide to SP94-AaLS via a thiolmaleimide Michael-type addition and introduction of anti-cancer drug bortezomib (BTZ). The cytoxicity of HepG2 cells treated with BTZ-Catechol-SP94-AaLS also increases in a dose-dependent manner, similar to that of free BTZ, while SP94-AaLS without BTZ has almost no cytotoxicity on the cells
additional information
homotopical sequence insertion into icosahedral lumazine synthase resulting in large particles. Mutations at the phosphate binding site Arg127 perturb enzymatic activity and also capsid assembly. The central channel of the pentameric building blocks appear significantly widened, indicating that the mode of interaction between the pentamer units and the topology of the subunit interfaces must have undergone significant changes, overview
additional information
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a 12-amino acid-long peptide at the C terminus of riboflavin synthase serves as a specific localization sequence responsible for targeting the guest to the protein compartment. Covalent fusion of this peptide tag to heterologous guest molecules leads to their internalization into lumazine synthase assemblies both in vivo and in vitro
additional information
a 12-amino acid-long peptide at the C terminus of riboflavin synthase serves as a specific localization sequence responsible for targeting the guest to the protein compartment. Covalent fusion of this peptide tag to heterologous guest molecules leads to their internalization into lumazine synthase assemblies both in vivo and in vitro
additional information
a circularly permuted variant of lumazine synthase affords versatile building blocks for the construction of nanocompartments that can be easily produced, tailored, and diversified. The topologically altered protein self-assembles into spherical and tubular cage structures with morphologies that can be controlled by the length of the linker connecting the native termini. Permutated lumazine synthase proteins integrate into wild-type and other engineered lumazine synthase assemblies by coproduction in Escherichia coli to form patchwork cages. This coassembly strategy enables encapsulation of guest proteins in the lumen, modification of the exterior through genetic fusion, and tuning of the size and electrostatics of the compartments
additional information
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a circularly permuted variant of lumazine synthase affords versatile building blocks for the construction of nanocompartments that can be easily produced, tailored, and diversified. The topologically altered protein self-assembles into spherical and tubular cage structures with morphologies that can be controlled by the length of the linker connecting the native termini. Permutated lumazine synthase proteins integrate into wild-type and other engineered lumazine synthase assemblies by coproduction in Escherichia coli to form patchwork cages. This coassembly strategy enables encapsulation of guest proteins in the lumen, modification of the exterior through genetic fusion, and tuning of the size and electrostatics of the compartments
additional information
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the C-terminal tail of ribiflavin synthase can act as an encapsulation tag capable of targeting other proteins to the lumazine synthase capsid interior. Fusion of to either the last 11 or the last 32 amino acids of riboflavin synthase, yields variant GFP11 or GFP32, respectively. After purification, lumazine synthase capsids that have been coproduced in bacteria with GFP11 and GFP32 are 15- and 6fold more fluorescent, respectively. GFP11 is localized within the lumazine synthase capsid. Fusing the last 11 amino acids of riboflavin synthase to the C-terminus of the Abrin A chain also leads to its encapsulation by lumazine synthase at a level similar to that of GFP11. Mild changes in pH and buffer identity trigger dissociation of the GFP11 guest
additional information
generation of a chimeric protein (BLS-FliC131) by fusing flagellin from Salmonella typhimurium in the N-termini of BLS. The fusion protein is recognized by anti-flagellin and anti-BLS antibodies, keeps the oligomerization capacity of BLS, without affecting the folding of the monomeric protein components. The thermal stability of each fusion partner is conserved. BLS-FliC131 retains the capacity of triggering TLR5. The humoral response against BLS elicited by BLS-FliC131 is stronger than the one elicited by equimolar amounts of BLS1FliC
additional information
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generation of a chimeric protein (BLS-FliC131) by fusing flagellin from Salmonella typhimurium in the N-termini of BLS. The fusion protein is recognized by anti-flagellin and anti-BLS antibodies, keeps the oligomerization capacity of BLS, without affecting the folding of the monomeric protein components. The thermal stability of each fusion partner is conserved. BLS-FliC131 retains the capacity of triggering TLR5. The humoral response against BLS elicited by BLS-FliC131 is stronger than the one elicited by equimolar amounts of BLS1FliC
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additional information
chloroplast transformation vector and analysis of transgene integration. The BLS scaffold is assessed for in plants for recombinant vaccine development by N-terminally fusing BLS to bovine rotavirus VP8d and expressing the resulting fusion (BLS-VP8d) in Nicotiana tabacum chloroplasts. BLS-VP8d remains soluble and stable during all stages of plant development and even in lyophilized leaves stored at room temperature
additional information
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construction of a chimera consisting of the B subunit of Shiga toxin type 2 and Brucella sp. lumazine synthase, BLS-Stx2B chimera, confers total protection against Shiga toxins in mice after immunization
additional information
for evaluation of the effectiveness of enzyme BLS as a preventive vaccine, C57BL/6J mice are immunized with BLS or BLS-OVA, subcutaneous inoculation with B16-OVA melanoma, and analysis of the effect induced by BLS in TLR4-expressing B16 melanoma cells. BLS or BLS-OVA induce a significant inhibition of tumor growth, survival is increased, and 50% of mice immunized with the highest dose of BLS do not develop visible tumors
additional information
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chloroplast transformation vector and analysis of transgene integration. The BLS scaffold is assessed for in plants for recombinant vaccine development by N-terminally fusing BLS to bovine rotavirus VP8d and expressing the resulting fusion (BLS-VP8d) in Nicotiana tabacum chloroplasts. BLS-VP8d remains soluble and stable during all stages of plant development and even in lyophilized leaves stored at room temperature
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