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Título: | New insights into the fructosyltransferase activity of Schwanniomyces occidentalis β-fructofuranosidase, emerging from nonconventional codon usage and directed mutation |
Autor: | Álvaro-Benito, Miguel CSIC ORCID; Abreu Felipe, Miguel A. de; García del Portillo, Francisco CSIC ORCID ; Sanz-Aparicio, J. CSIC ORCID; Fernández Lobato, María CSIC ORCID | Fecha de publicación: | 2010 | Editor: | American Society for Microbiology | Citación: | Applied and Environmental Microbiology 76(22): 7491-7499 (2010) | Resumen: | 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 (kcat/K m) of the enzyme (approximately 1,000-fold), but surprisingly, its transferase activity is enhanced by almost 3-fold, as is the enzymeś 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. Copyright © 2010, American Society for Microbiology. All Rights Reserved. | URI: | http://hdl.handle.net/10261/77458 | DOI: | 10.1128/AEM.01614-10 | Identificadores: | doi: 10.1128/AEM.01614-10 issn: 0099-2240 e-issn: 1098-5336 |
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