Substrates: the asymmetric reductase intercepts acyclic imino acids produced in situ by a partner oxidase (Ind4 from Paenibacillus sp.) in a coupled assay, overview. Nonenzymatic hydrolysis of acyclic imino acids gives compounds (4E)-5-carbamimidamido-2-oxopent-4-enoic acid and 5-carbamimidamido-2-oxopentanoic acid. Bsp5 fails to reduce 5-carbamimidamido-2-oxopentanoic acid, and incubation of Bsp5 with 2-methylpyrroline also results in no reaction. Bsp5 lacks imine reducing activity on cyclic substrates. LC-MS analysis of the molecules Products: -
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified enzyme Bsp5 in complex with D-arginine and coenzyme NADPH, hanging drop vapour diffusion, mixing of 800 nl of 10 mg/m protein in 20 mM HEPES, 50 mM NaCl, pH 7.5, 5 mM D-Arg, and 5 mM NADPH, with 800 nl of reservoir solution containing 20% w/v PEG 3350 and 0.2 M sodium tartrate dibasic, equilibration against 0.05 ml of reservoir solution, room temperature, 10 days, method optimization, X-ray diffraction structure determination and analysis at 1.6 A resolution, molecular replacement, modeling
site-directed mutagenesis, the mutant shows highly reduced activity with substrate (4E)-5-carbamimidamido-2-iminopent-4-enoic acid, but unaltered activity with 5-carbamimidamido-2-iminopentanoic acid compared to wild-type
site-directed mutagenesis, the mutant shows highly reduced activity with substrate (4E)-5-carbamimidamido-2-iminopent-4-enoic acid, but increased activity with 5-carbamimidamido-2-iminopentanoic acid compared to wild-type
site-directed mutagenesis, the mutant shows reduced activity with substrates (4E)-5-carbamimidamido-2-iminopent-4-enoic acid and 5-carbamimidamido-2-iminopentanoic acid compared to wild-type
site-directed mutagenesis, the mutant shows highly reduced activity with substrate (4E)-5-carbamimidamido-2-iminopent-4-enoic acid, but unaltered activity with 5-carbamimidamido-2-iminopentanoic acid compared to wild-type
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, gel filtration, and ultrafiltration
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene bsp5, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant overexpression of His6-tagged wild-type and mutant enzymes from pUC57-Bsp5 vector in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH5alpha
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