Molecular characterization of inulosucrase from Leuconostoc citreum: a fructosyltransferase within a glucosyltransferase.
The gene coding for inulosucrase in Leuconostoc citreum CW28, islA, was cloned, sequenced, and expressed in Escherichia coli. The recombinant enzyme catalyzed inulin synthesis from sucrose like the wild-type enzyme. Inulosucrase presents an unusual structure: its N-terminal region is similar to the variable region of glucosyltransferases, its catalytic domain is similar to fructosyltransferases from various microorganisms, and its C-terminal domain presents similarity to the glucan binding domain from alternansucrase, a glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355. From sequence comparison, it was found that this fructosyltransferase is a natural chimeric enzyme resulting from the substitution of the catalytic domain of alternansucrase by a fructosyltransferase. Two different forms of the islA gene truncated in the C-terminal glucan binding domain were successfully expressed in E. coli and retained their ability to synthesize inulin but lost thermal stability. This is the first report of an inulosucrase bearing structural features of both glucosyltransferases and fructosyltransferases.
Olivares-Illana
Vanesa
V
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca Morelo, Mexico.
López-Munguía
Agustín
A
Olvera
Clarita
C
D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism; D005936:Glucans / Q000737:chemistry; D005964:Glucosyltransferases / Q000737:chemistry; D006602:Hexosyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D007444:Inulin / Q000096:biosynthesis; D007934:Leuconostoc / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D001426:Bacterial Proteins
D005936:Glucans; D005964:Glucosyltransferases; D006602:Hexosyltransferases; D007444:Inulin; D011994:Recombinant Proteins; C021451:inulosucrase; D001426:Bacterial Proteins