Newly identified essential amino acid residues affecting ¦¤⁸-sphingolipid desaturase activity revealed by site-directed mutagenesis

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• 作者：Shu-Fen  ; Li^a ;   ; ^d ;   ; ¹ ;   ; ^{sfli@genetics.ac.cn} ; Li-Ying  ; Song^a ;   ; ¹ ;   ; ^{lysong@genetics.ac.cn} ; Guo-Jun  ; Zhang^b ;   ; ^{zgj003@xxmu.edu.cn} ; Wei-Bo  ; Yin^a ;   ; ^{wbyin@genetics.ac.cn} ; Yu-Hong  ; Chen^a ;   ; ^{yhchen@genetics.ac.cn} ; Richard R.-C.  ; Wang^c ;   ; ^{Richard.Wang@ARS.USDA.gov} ; Zan-Min  ; Hu^a ;   ; ^{zmhu@genetics.ac.cn}
• 关键词：¦¤⁸-sphingolipid desaturase ; Site-directed mutagenesis ; Yeast expression ; Desaturation activity
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：2011
• 出版时间：9 December 2011
• 年：2011
• 卷：416
• 期：1-2
• 页码：165-171
• 全文大小：793 K

文摘

In order to identify amino acid residues crucial for the enzymatic activity of ¦¤⁸-sphingolipid desaturases, a sequence comparison was performed among ¦¤⁸-sphingolipid desaturases and ¦¤⁶-fatty acid desaturases from various plants. In addition to the known conserved cytb₅ (cytochrome b₅) HPGG motif and three conserved histidine boxes, they share additional 15 completely conserved residues. A series of site-directed mutants were generated using our previously isolated ¦¤⁸-sphingolipid desaturase gene from Brassica rapa to evaluate the importance of these residues to the enzyme function. The mutants were functionally characterized by heterologous expression in yeast, allowing the identification of the products of the enzymes. The results revealed that residues H63, N203, D208, D210, and G368 were obligatorily required for the enzymatic activity, and substitution of the residues F59, W190, W345, L369 and Q372 markedly decreased the enzyme activity. Among them, replacement of the residues W190, L369 and Q372 also has significant influence on the ratio of the two enzyme products. Information obtained in this work provides the molecular basis for the ¦¤⁸-sphingolipid desaturase activity and aids in our understanding of the structure¨Cfunction relationships of the membrane-bound desaturases.