New insights into the fructosyltransferase activity of Schwanniomyces occidentalis ß-fructofuranosidase, emerging from nonconventional codon usage and directed mutation.
Schwanniomyces occidentalis β-fructofuranosidase (Ffase) releases β-fructose from the nonreducing ends of β-fructans and synthesizes 6-kestose and 1-kestose, both considered prebiotic fructooligosaccharides. Analyzing the amino acid sequence of this protein revealed that it includes a serine instead of a leucine at position 196, caused by a nonuniversal decoding of the unique mRNA leucine codon CUG. Substitution of leucine for Ser196 dramatically lowers the apparent catalytic efficiency (k(cat)/K(m)) of the enzyme (approximately 1,000-fold), but surprisingly, its transferase activity is enhanced by almost 3-fold, as is the enzymes' specificity for 6-kestose synthesis. The influence of 6 Ffase residues on enzyme activity was analyzed on both the Leu196/Ser196 backgrounds (Trp47, Asn49, Asn52, Ser111, Lys181, and Pro232). Only N52S and P232V mutations improved the transferase activity of the wild-type enzyme (about 1.6-fold). Modeling the transfructosylation products into the active site, in combination with an analysis of the kinetics and transfructosylation reactions, defined a new region responsible for the transferase specificity of the enzyme.
Alvaro-Benito
Miguel
M
Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular (UAM-CSIC), Universidad Autónoma Madrid, Cantoblanco, 28049 Madrid, Spain.
de Abreu
Miguel
M
Portillo
Francisco
F
Sanz-Aparicio
Julia
J
Fernández-Lobato
María
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D003062:Codon / Q000235:genetics*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D005630:Fructans / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D004718:Saccharomycetales / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D014312:Trisaccharides / Q000378:metabolism; D043324:beta-Fructofuranosidase / Q000235:genetics* / Q000378:metabolism*
D003062:Codon; D004271:DNA, Fungal; D005630:Fructans; D014312:Trisaccharides; C072433:1-kestose; C073432:6-kestose; D043324:beta-Fructofuranosidase