Taxonomic group: plant / Streptophyta
(Phylum: Streptophyta)
NCBI PubMed ID: 10748038Publication DOI: 10.1074/jbc.M001609200Journal NLM ID: 2985121RPublisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
Correspondence: Esen A <aevatan
vt.edu>
Institutions: Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, USA, Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, USA
The maize β-glucosidase isozyme Glu1 hydrolyzes a broad spectrum of substrates in addition to its natural substrate DIMBOAGlc (2-O-β-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one), whereas the sorghum β-glucosidase isozyme Dhr1 hydrolyzes exclusively its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-β-D-glucose). To study the mechanism of substrate specificity further, eight chimeric β-glucosidases were constructed by replacing peptide sequences within the C-terminal region of Glu1 with the homologous peptide sequences of Dhr1 or vice versa, where the two enzymes differ by 4 to 22 amino acid substitutions, depending on the length of the swapped regions. Five Glu1/Dhr1 chimeras hydrolyzed substrates that are hydrolyzed by both parental enzymes, including dhurrin, which is not hydrolyzed by Glu1. In contrast, three Dhr1/Glu1 chimeras hydrolyzed only dhurrin but with lower catalytic efficiency than Dhr1. Additional domain-swapping within the C-terminal domain of Glu1 showed that replacing the peptide (466)FAGFTERY(473) of Glu1 with the homologous peptide (462)SSGYTERF(469) of Dhr1 or replacing the peptide (481)NNNCTRYMKE(490) in Glu1 with the homologous peptide (477)ENGCERTMKR(486) of Dhr1 was sufficient to confer to Glu1 the ability to hydrolyze dhurrin. Data from various reciprocal chimeras, sequence comparisons, and homology modeling suggest that the Dhr1-specific Ser-462-Ser-463 and Phe-469 play a key role in dhurrin hydrolysis. Similar data suggest that DIMBOAGlc hydrolysis determinants are not located within the extreme 47-amino acid-long C-terminal domain of Glu1.
glucoside, β-glucosidases, sorghum, maize, DIMBOA
Structure type: monomer
Location inside paper: p. 20003, Fig. 1, A, dhurrin
Trivial name: (S)-dhurrin, dhurrin
Compound class: glycoside, cyanogenic glycoside
Contained glycoepitopes: IEDB_142488,IEDB_146664,IEDB_983931,SB_192
Methods: PCR, SDS-PAGE, DNA techniques, inhibition studies, TLC, extraction, column chromatography, enzymatic assay, precipitation, centrifugation, DNA expression
Enzymes that release or process the structure: dhurrinase, sorghum β-glucosidase
Related record ID(s): 65162
NCBI Taxonomy refs (TaxIDs): 4577,
4558
Show glycosyltransferases
There is only one chemically distinct structure:
Taxonomic group: plant / Streptophyta
(Phylum: Streptophyta)
NCBI PubMed ID: 10748038Publication DOI: 10.1074/jbc.M001609200Journal NLM ID: 2985121RPublisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
Correspondence: Esen A <aevatan
vt.edu>
Institutions: Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, USA, Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, USA
The maize β-glucosidase isozyme Glu1 hydrolyzes a broad spectrum of substrates in addition to its natural substrate DIMBOAGlc (2-O-β-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one), whereas the sorghum β-glucosidase isozyme Dhr1 hydrolyzes exclusively its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-β-D-glucose). To study the mechanism of substrate specificity further, eight chimeric β-glucosidases were constructed by replacing peptide sequences within the C-terminal region of Glu1 with the homologous peptide sequences of Dhr1 or vice versa, where the two enzymes differ by 4 to 22 amino acid substitutions, depending on the length of the swapped regions. Five Glu1/Dhr1 chimeras hydrolyzed substrates that are hydrolyzed by both parental enzymes, including dhurrin, which is not hydrolyzed by Glu1. In contrast, three Dhr1/Glu1 chimeras hydrolyzed only dhurrin but with lower catalytic efficiency than Dhr1. Additional domain-swapping within the C-terminal domain of Glu1 showed that replacing the peptide (466)FAGFTERY(473) of Glu1 with the homologous peptide (462)SSGYTERF(469) of Dhr1 or replacing the peptide (481)NNNCTRYMKE(490) in Glu1 with the homologous peptide (477)ENGCERTMKR(486) of Dhr1 was sufficient to confer to Glu1 the ability to hydrolyze dhurrin. Data from various reciprocal chimeras, sequence comparisons, and homology modeling suggest that the Dhr1-specific Ser-462-Ser-463 and Phe-469 play a key role in dhurrin hydrolysis. Similar data suggest that DIMBOAGlc hydrolysis determinants are not located within the extreme 47-amino acid-long C-terminal domain of Glu1.
glucoside, β-glucosidases, sorghum, maize, DIMBOA
Structure type: monomer
Location inside paper: p. 20003, Fig. 1, B
Trivial name: DIMBOA glucoside
Compound class: glycoside
Contained glycoepitopes: IEDB_142488,IEDB_146664,IEDB_983931,SB_192
Methods: PCR, SDS-PAGE, DNA techniques, inhibition studies, TLC, extraction, column chromatography, enzymatic assay, precipitation, centrifugation, DNA expression
Enzymes that release or process the structure: DIMBOA-glucosidase, maize β-glucosidase
Related record ID(s): 65161
NCBI Taxonomy refs (TaxIDs): 4577,
4558
Show glycosyltransferases
There is only one chemically distinct structure:
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