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KMID : 1094720140190050798
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Biotechnology and Bioprocess Engineering
2014 Volume.19 No. 5 p.798 ~ p.802
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Effect of His-tag location on the catalytic activity of 3-hydroxybutyrate dehydrogenase
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Yeon Young-Joo
Park Hyun-June
Park Hyung-Yeon
Yoo Young-Je
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Abstract
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The effect of hexahistidine-tag (His-tag) location at either the C or N-terminus on the catalytic activity of 3-hydroxybutyrate dehydrogenase (3HBDH) from Alcaligenes faecalis was studied. The kinetic parameters of 3HBDHs with C and N-terminal His-tags were investigated, and the enzyme with an N-terminal His-tag was found to have approximately 1,200-fold higher catalytic efficiency than its C-terminal counterpart. Furthermore, the effect of His-tag location on the catalytic activity of 3 engineered variants of 3HBDH that were previously developed for the conversion of levulinic acid to 4-hydroxyvaleric acid was also investigated. All of the N-terminal variants exhibited higher catalytic efficiency for levulinic acid than did the C-terminal counterparts. The structural basis of the His-tag effect was studied by investigating the structure of 3HBDH obtained from in silico His-tag modification, and the results revealed that the modification of the C-terminal structure could deform the hinge region of the active site entry loop, disrupting the catalytic motion of the enzyme. In contrast, due to the location of the N-terminus far from the active site of the enzyme, the catalytic activity of the enzyme was not severely affected by the N-terminal His-tag.
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KEYWORD
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location of his-tag, catalytic activity, 3-Hydroxybutyrate dehydrogenase, entry loop, hydrogen bond
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