Reactivity of sorbose dehydrogenase from <i>Sinorhizobium</i> sp. 97507 for 1,5-anhydro-<scp>d</scp>-glucitol

Araki, Toshimitsu; Nakatsuka, Tomoko; Kobayashi, Fuminao; Watanabe-Ishimaru, Emi; Sanada, Hirokazu; Tamura, Takashi; Inagaki, Kenji

doi:10.1080/09168451.2015.1012148

Cited by 2 publications

(1 citation statement)

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“…Among them, the A551N and A551L mutants were less active towards glucose and more specific for 1,5-AG, relative to WT. Although their catalytic efficiency was decreased significantly and may be too low for clinical application, 22) we believe that they should be useful as leads for further optimization to obtain more active and 1,5-AG-specific enzymes by means of random mutagenesis 23) 24) 25) and high-throughput screening, 26) 27) paving the way for clinical application.…”

Section: Resultsmentioning

confidence: 97%

Single Amino Acid Mutation of Pyranose 2-Oxidase Results in Increased Specificity for Diabetes Biomarker 1,5-Anhydro-D-Glucitol

2020

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Pyranose 2-oxidases catalyze the oxidation of various pyranose sugars at the C2 position. However, their potential application for detecting sugars other than glucose in blood is hindered by relatively high activity towards glucose. In this study, in order to find a mutant enzyme with enhanced specificity for 1,5-anhydro-D-glucitol (1,5-AG), which is a biomarker for diabetes mellitus, we conducted site-directed mutagenesis of pyranose 2-oxidase from the basidiomycete Phanerochaete chrysosporium ( Pc POX). Considering the three-dimensional structure of the substrate-binding site of Pc POX and the structural difference between glucose and 1,5-AG, we selected alanine 551 of Pc POX as a target residue for mutation. Kinetic studies of the 19 mutants of Pc POX expressed as recombinant proteins in E. coli revealed that the ratio of k cat / K m for 1,5-AG to k cat / K m for glucose was three times higher for the A551L mutant than for wild-type Pc POX. Although the A551L mutant has lower specific activity towards each substrate than the wild-type enzyme, its increased specificity for 1,5-AG makes it a promising lead for the development of POX-based 1,5-AG detection systems.

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