Kinetic studies of levansucrase of Bacillus subtilis
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Foundational kinetic characterization of levansucrase
"Kinetic studies of levansucrase of Bacillus subtilis"
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Established dual hydrolase/transferase activity depending on sucrose concentration
"Kinetic studies of levansucrase of Bacillus subtilis"
Structural framework of fructosyl transfer in Bacillus subtilis levansucrase
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First crystal structure of B. subtilis levansucrase at 1.50 angstroms
"The crystal structure of Bacillus subtilis levansucrase, determined to a resolution of 1.5 A, shows a rare five-fold beta-propeller topology with a deep, negatively charged central pocket"
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Identified catalytic residues Asp86 (nucleophile), Asp247 (transition state stabilizer), Glu342 (acid/base)
"three conserved acidic side chains in the central pocket are critical for catalysis, and presumably function as nucleophile (Asp86) and general acid (Glu342), or stabilize the transition state (Asp247)"
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Characterized Ca2+ binding sites
"The crystal structure of Bacillus subtilis levansucrase, determined to a resolution of 1.5 A"
Selected mutations in Bacillus subtilis levansucrase semi-conserved regions affecting its biochemical properties
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Mutagenesis studies characterizing active site residues
"From a multiple sequence analysis of GH68 family proteins, nine residues were selected and their role in acceptor and product specificity, as well as in biochemical Bacillus subtilis LS properties, was investigated"
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Km of 8 mM for sucrose
"S164 is an important residue to maintain the nucleophile position in the active site"
Implications of the mutation S164A on Bacillus subtilis levansucrase product specificity and insights into protein interactions acting upon levan synthesis.
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Detailed kinetic analysis of hydrolysis vs transfructosylation
"Mutation S164A largely affects the transfructosylation properties of Bacillus subtilis levansucrase (SacB). The variant uses acceptors such as glucose and short levans with an average molecular weight of 7.6 kDa more efficiently than SacB"
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S164A mutation increases transfructosylation activity
"A 3-fold increase in blasto-oligosaccharides yield is provoked by the modified interplay between the variant and glucose"
The molecular basis of the nonprocessive elongation mechanism in levansucrases
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Crystal structure with levan hexasaccharides
"we determined the crystallographic structure of Bacillus subtilis LS (SacB) in complex with a levan-type fructooligosaccharide and utilized site-directed mutagenesis to identify residues involved in substrate binding"
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Molecular basis for nonprocessive elongation mechanism
"Our results clarify, for the first time, the interaction of the enzyme with an acceptor/product oligosaccharide and elucidate the molecular basis of the nonprocessive levan elongation mechanism of LSs"
Characterization of the precursor form of the exocellular levansucrase from Bacillus subtilis
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Characterized signal peptide and secretion
"The existence of a precursor form of the enzyme of MW 53000 was also demonstrated and confirmed by the DNA sequence corresponding to the NH2 terminal region of the protein"
yveB, encoding endolevanase LevB, is part of the sacB-yveB-yveA levansucrase tricistronic operon in Bacillus subtilis
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sacB is part of a tricistronic operon with yveB (endolevanase) and yveA
"sacB, encoding levansucrase, is the proximal gene of a sucrose-inducible operon that includes the two other genes"
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Induced by sucrose
"Transcription of sacB, yveB and yveA, three clustered genes on the Bacillus subtilis chromosome, is simultaneously induced by sucrose"
Gene Ontology annotation through association of InterPro records with GO terms
Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword mapping
Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping
Combined Automated Annotation using Multiple IEA Methods
Deep research report on sacB levansucrase
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Comprehensive review of levansucrase biology from recent literature (2023-2024)
"sacB encodes levansucrase (SacB) from Bacillus subtilis (strain 168); UniProt P05655"
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Details on operon organization and secretion
"SacB possesses an N-terminal Sec-type signal peptide and is secreted extracellularly in Gram-positive bacteria"
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GH68 levansucrases use retaining double-displacement mechanism with 5-bladed beta-propeller fold
"GH68 levansucrases operate via a retaining double-displacement mechanism"
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Ca2+ and other divalent metals can stimulate fructosylation
"Metal-binding loop in some levansucrases; certain divalent ions (e.g., Ca2+) can stimulate fructosylation/levan synthesis by allosteric effects"