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Enzymatic Production of Non‐Natural Nucleoside‐5′‐Monophosphates by a Thermostable Uracil Phosphoribosyltransferase
Author(s) -
del Arco Jon,
Acosta Javier,
Pereira Humberto M.,
Perona Almudena,
Lokanath Neratur K.,
Kunishima Naoki,
FernándezLucas Jesús
Publication year - 2018
Publication title -
chemcatchem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.497
H-Index - 106
eISSN - 1867-3899
pISSN - 1867-3880
DOI - 10.1002/cctc.201701223
Subject(s) - chemistry , uracil , homotetramer , biocatalysis , thermus thermophilus , enzyme , substrate (aquarium) , nucleoside , biochemistry , combinatorial chemistry , escherichia coli , biology , reaction mechanism , catalysis , dna , ecology , protein subunit , gene
The use of enzymes as biocatalysts applied to synthesis of modified nucleoside‐5′‐monophosphates (NMPs) is an interesting alternative to traditional multistep chemical methods which offers several advantages, such as stereo‐, regio‐, and enantioselectivity, simple downstream processing, and mild reaction conditions. Herein we report the recombinant expression, production, and purification of uracil phosphoribosyltransferase from Thermus themophilus HB8 ( Tt UPRT). The structure of Tt UPRT has been determined by protein crystallography, and its substrate specificity and biochemical characteristics have been analyzed, providing new structural insights into the substrate‐binding mode. Biochemical characterization of the recombinant protein indicates that the enzyme is a homotetramer, with activity and stability across a broad range of temperatures (50–80 °C), pH (5.5–9) and ionic strength (0–500 m m NaCl). Surprisingly, Tt UPRT is able to recognize several 5 and 6‐substituted pyrimidines as substrates. These experimental results suggest Tt UPRT could be a valuable biocatalyst for the synthesis of modified NMPs.