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Tyl1a, a TDP‐6‐deoxy‐ D ‐ xylo ‐4‐hexulose 3,4‐isomerase from Streptomyces fradiae : Structure Prediction, Mutagenesis and Solvent Isotope Incorporation Experiments to Investigate Reaction Mechanism
Author(s) -
Tello Mónica,
Rejzek Martin,
Wilkinson Barrie,
Lawson David M.,
Field Robert A.
Publication year - 2008
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200800021
Subject(s) - deprotonation , chemistry , isomerase , protonation , stereochemistry , biochemistry , protein engineering , mutagenesis , isomerization , nucleotide , saturated mutagenesis , site directed mutagenesis , directed evolution , glycosylation , enzyme , mutant , organic chemistry , ion , gene , catalysis
Understanding the structure and mechanism of sugar nucleotide processing enzymes is invaluable in the generation of designer enzymes for biotransformation, for instance, in connection with engineering antibiotic glycosylation. In this study, homology modelling and mechanistic comparison to the structurally related RmlC epimerase family has been used to identify and assign functions to active‐site residues in the Tyl1a‐catalysed keto‐sugar nucleotide isomerisation process. Tyl1a His63 is implicated as the base that initiates the isomerisation process by substrate C‐3 deprotonation, with Arg109 stabilising the resulting enolate. Subsequent O‐3 deprotonation (potentially by His65) and C‐4 protonation (potentially by Tyr49) complete the isomerisation process.