New insights into the fructosyltransferase activity of Schwanniomyces occidentalis ß-fructofuranosidase, emerging from nonconventional codon usage and directed mutation.

ABSTRACT: Schwanniomyces occidentalis ?-fructofuranosidase (Ffase) releases ?-fructose from the nonreducing ends of ?-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.

SUBMITTER: Alvaro-Benito M 

PROVIDER: S-EPMC2976189 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature