Structural and functional features of fructansucrases present in Leuconostoc mesenteroides ATCC 8293.
Glycosyltransferases produced by Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 (equivalent to NRRL B-1118) were identified. Two glucansucrases and one fructansucrases were observed in batch culture while levC and levL genes, corresponding to two fructansucrases, were isolated from information obtained from the released draft sequence of this Leuconostoc strain genome and cloned in Escherichia coli. The recombinant enzymes were shown to be fructansucrases producing a polymer identified by NMR as levan, confirming our recent report stating that these are also mosaic levansucrases bearing structural features of glucansucrases in the amino and carboxy terminal regions, as is also the case of inulosucrase (IslA) from Leuconostoc citreum CW28 and levansucrase (LevS) from L. mesenteroides NRRL B-512F. The recombinant levansucrase LevC was purified and characterized in terms of pH, temperature, and kinetic properties. The enzyme exhibits Michaelis-Menten kinetic properties with a K(m) = 27.3 mM and a k(cat) = 282.9 s(-1). This levansucrase behaves mainly as a transferase as only 30% of the substrate is hydrolyzed in a wide range of sucrose concentrations, with higher hydrolytic activities at low substrate concentrations. With this report we experimentally confirm the unusual structural pattern displayed by fructansucrases present in Leuconostoc species that group as a novel sub family of fructansucrases.
Olvera
Clarita
C
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001 Col. Chamilpa, Cuernavaca, Morelos, 62250, Mexico.
Centeno-Leija
Sara
S
López-Munguía
Agustín
A
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism; D019295:Computational Biology; D004591:Electrophoresis, Polyacrylamide Gel; D005630:Fructans / Q000378:metabolism; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D037102:Lectins / Q000378:metabolism; D007934:Leuconostoc / Q000201:enzymology* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011887:Raffinose / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism; D013696:Temperature; D001426:Bacterial Proteins
D002352:Carrier Proteins; D005630:Fructans; D006602:Hexosyltransferases; D037102:Lectins; D011887:Raffinose; D013395:Sucrose; C037937:glucan-binding proteins; C072599:levan; C021513:levansucrase; C021451:inulosucrase; D001426:Bacterial Proteins