Enhanced Fructose Oxidase Activity in a Galactose Oxidase Variant
Galactose oxidase (GO; EC 1.1.3.9) catalyses the oxidation of a wide range of primary alcohols including mono-, oligo- and polysaccharides. High-resolution structures have been determined for GO, but no structural information is available for the enzyme with bound substrate or inhibitor. Previously,...
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| Veröffentlicht in: | Chembiochem : a European journal of chemical biology 2004-07, Vol.5 (7), p.972-979 |
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| container_title | Chembiochem : a European journal of chemical biology |
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| creator | Deacon, Sarah E Mahmoud, Khaled Spooner, R. Kate Firbank, Susan J Knowles, Peter F Phillips, Simon E.V McPherson, Michael J |
| description | Galactose oxidase (GO; EC 1.1.3.9) catalyses the oxidation of a wide range of primary alcohols including mono-, oligo- and polysaccharides. High-resolution structures have been determined for GO, but no structural information is available for the enzyme with bound substrate or inhibitor. Previously, computer-aided docking experiments have been used to develop a plausible model for interactions between GO and the D-galactose substrate. Residues implicated in such interactions include Arg330, Gln406, Phe464, Phe194 and Trp290. In the present study we describe an improved expression system for recombinant GO in the methylotrophic yeast Pichia pastoris. We use this system to express variant proteins mutated at Arg330 and Phe464 to explore the substrate binding model. We also demonstrate that the Arg330 variants display greater fructose oxidase activity than does wild-type GO. |
| doi_str_mv | 10.1002/cbic.200300810 |
| format | Article |
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Kate ; Firbank, Susan J ; Knowles, Peter F ; Phillips, Simon E.V ; McPherson, Michael J</creator><creatorcontrib>Deacon, Sarah E ; Mahmoud, Khaled ; Spooner, R. Kate ; Firbank, Susan J ; Knowles, Peter F ; Phillips, Simon E.V ; McPherson, Michael J</creatorcontrib><description>Galactose oxidase (GO; EC 1.1.3.9) catalyses the oxidation of a wide range of primary alcohols including mono-, oligo- and polysaccharides. High-resolution structures have been determined for GO, but no structural information is available for the enzyme with bound substrate or inhibitor. Previously, computer-aided docking experiments have been used to develop a plausible model for interactions between GO and the D-galactose substrate. Residues implicated in such interactions include Arg330, Gln406, Phe464, Phe194 and Trp290. In the present study we describe an improved expression system for recombinant GO in the methylotrophic yeast Pichia pastoris. We use this system to express variant proteins mutated at Arg330 and Phe464 to explore the substrate binding model. We also demonstrate that the Arg330 variants display greater fructose oxidase activity than does wild-type GO.</description><identifier>ISSN: 1439-4227</identifier><identifier>EISSN: 1439-7633</identifier><identifier>DOI: 10.1002/cbic.200300810</identifier><identifier>PMID: 15239055</identifier><language>eng</language><publisher>Weinheim: Wiley-VCH Verlag</publisher><subject>copper enzymes ; Crystallography, X-Ray ; enzyme catalysis ; fructose ; Fructose - metabolism ; Galactose - metabolism ; Galactose Oxidase - chemistry ; Galactose Oxidase - genetics ; Galactose Oxidase - metabolism ; Mutation ; oxidases ; Oxidation-Reduction ; Pichia - genetics ; protein engineering ; Recombinant Proteins - genetics ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - metabolism ; Structure-Activity Relationship ; Substrate Specificity ; Transformation, Genetic</subject><ispartof>Chembiochem : a European journal of chemical biology, 2004-07, Vol.5 (7), p.972-979</ispartof><rights>Copyright © 2004 WILEY‐VCH Verlag GmbH & Co. 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Kate</creatorcontrib><creatorcontrib>Firbank, Susan J</creatorcontrib><creatorcontrib>Knowles, Peter F</creatorcontrib><creatorcontrib>Phillips, Simon E.V</creatorcontrib><creatorcontrib>McPherson, Michael J</creatorcontrib><title>Enhanced Fructose Oxidase Activity in a Galactose Oxidase Variant</title><title>Chembiochem : a European journal of chemical biology</title><addtitle>ChemBioChem</addtitle><description>Galactose oxidase (GO; EC 1.1.3.9) catalyses the oxidation of a wide range of primary alcohols including mono-, oligo- and polysaccharides. High-resolution structures have been determined for GO, but no structural information is available for the enzyme with bound substrate or inhibitor. Previously, computer-aided docking experiments have been used to develop a plausible model for interactions between GO and the D-galactose substrate. Residues implicated in such interactions include Arg330, Gln406, Phe464, Phe194 and Trp290. In the present study we describe an improved expression system for recombinant GO in the methylotrophic yeast Pichia pastoris. We use this system to express variant proteins mutated at Arg330 and Phe464 to explore the substrate binding model. We also demonstrate that the Arg330 variants display greater fructose oxidase activity than does wild-type GO.</description><subject>copper enzymes</subject><subject>Crystallography, X-Ray</subject><subject>enzyme catalysis</subject><subject>fructose</subject><subject>Fructose - metabolism</subject><subject>Galactose - metabolism</subject><subject>Galactose Oxidase - chemistry</subject><subject>Galactose Oxidase - genetics</subject><subject>Galactose Oxidase - metabolism</subject><subject>Mutation</subject><subject>oxidases</subject><subject>Oxidation-Reduction</subject><subject>Pichia - genetics</subject><subject>protein engineering</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Recombinant Proteins - metabolism</subject><subject>Structure-Activity Relationship</subject><subject>Substrate Specificity</subject><subject>Transformation, Genetic</subject><issn>1439-4227</issn><issn>1439-7633</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkElPwzAQRi0EYilcOUJO3FLGS-zmWKK2IJVFKtvNchwHDGlS7ATovydVKpYTpxlp3vdJ8xA6xNDHAORUp1b3CQAFGGDYQLuY0TgUnNLN9c4IETtoz_sXAIg5xdtoB0eExhBFu2g4Kp9VqU0WjF2j68qb4PrTZqqdQ13bd1svA1sGKpioQv293ytnVVnvo61cFd4crGcP3Y1Ht8l5OL2eXCTDaagZoxAqEQ8IFywDodOUCKrZgOYcZzSHNE8xsNiw9gmCI56DziDPVMqjOOOUCcMx7aGTrnfhqrfG-FrOrdemKFRpqsZLzvmARIy2YL8Dtau8dyaXC2fnyi0lBrmSJlfS5Le0NnC0bm7Sucl-8LWlFog74MMWZvlPnUzOLpLf5WGXtb42n99Z5V4lF1RE8uFqIh_5_eXV7GYsk5Y_7vhcVVI9Oevl3YwApoBjRjAh9Av-QY9f</recordid><startdate>20040705</startdate><enddate>20040705</enddate><creator>Deacon, Sarah E</creator><creator>Mahmoud, Khaled</creator><creator>Spooner, R. 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Kate ; Firbank, Susan J ; Knowles, Peter F ; Phillips, Simon E.V ; McPherson, Michael J</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4430-a7982674d07cbb273c483f61d3f0bfb1049e40082156f0cd0fdab659d6347e613</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>copper enzymes</topic><topic>Crystallography, X-Ray</topic><topic>enzyme catalysis</topic><topic>fructose</topic><topic>Fructose - metabolism</topic><topic>Galactose - metabolism</topic><topic>Galactose Oxidase - chemistry</topic><topic>Galactose Oxidase - genetics</topic><topic>Galactose Oxidase - metabolism</topic><topic>Mutation</topic><topic>oxidases</topic><topic>Oxidation-Reduction</topic><topic>Pichia - genetics</topic><topic>protein engineering</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Recombinant Proteins - metabolism</topic><topic>Structure-Activity Relationship</topic><topic>Substrate Specificity</topic><topic>Transformation, Genetic</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Deacon, Sarah E</creatorcontrib><creatorcontrib>Mahmoud, Khaled</creatorcontrib><creatorcontrib>Spooner, R. 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Previously, computer-aided docking experiments have been used to develop a plausible model for interactions between GO and the D-galactose substrate. Residues implicated in such interactions include Arg330, Gln406, Phe464, Phe194 and Trp290. In the present study we describe an improved expression system for recombinant GO in the methylotrophic yeast Pichia pastoris. We use this system to express variant proteins mutated at Arg330 and Phe464 to explore the substrate binding model. We also demonstrate that the Arg330 variants display greater fructose oxidase activity than does wild-type GO.</abstract><cop>Weinheim</cop><pub>Wiley-VCH Verlag</pub><pmid>15239055</pmid><doi>10.1002/cbic.200300810</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | copper enzymes Crystallography, X-Ray enzyme catalysis fructose Fructose - metabolism Galactose - metabolism Galactose Oxidase - chemistry Galactose Oxidase - genetics Galactose Oxidase - metabolism Mutation oxidases Oxidation-Reduction Pichia - genetics protein engineering Recombinant Proteins - genetics Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism Structure-Activity Relationship Substrate Specificity Transformation, Genetic |
| title | Enhanced Fructose Oxidase Activity in a Galactose Oxidase Variant |
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