The aglycone specificity-determining sites are different in 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA)-glucosidase (maize β-glucosidase) and dhurrinase (sorghum β-glucosidase)

被引:46
作者
Cicek, M
Blanchard, D
Bevan, DR
Esen, A [1 ]
机构
[1] Virginia Polytech Inst & State Univ, Dept Biol, Blacksburg, VA 24061 USA
[2] Virginia Polytech Inst & State Univ, Dept Biochem, Blacksburg, VA 24061 USA
关键词
D O I
10.1074/jbc.M001609200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The maize beta-glucosidase isozyme Glu1 hydrolyzes a broad spectrum of substrates in addition to its natural substrate DIMBOAGlc (2-O-beta-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one), whereas the sorghum beta-glucosidase isozyme Dhr1 hydrolyzes exclusively its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-beta-D-glucose). To study the mechanism of substrate specificity further, eight chimeric beta-glucosidases were constructed by replacing peptide sequences within the C-terminal region of Glu1 with the homologous peptide sequences of Dhr1 or vice verse, 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 (NNNCTRYMKE490)-N-481 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.
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页码:20002 / 20011
页数:10
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