請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/21828
標題: Biochemical characterization of N-acylamino acid racemase from Deinococcus radiodurans BCRC12827 and its application in the enantioselective synthesis of L-homophenylalanine
Deinococcus radiodurans BCRC12827的N-acylamino acid racemase酵素生化性質之分析及應用此酵素參與光學選擇性合成L-homophenylalanine
作者: 許世光
Hsu, Shih-Kuang
關鍵字: N-acylamino acid racemase
Muconate lactonizing enzyme
o-succinylbenzoate synthase
L-homophenylalanine
出版社: 分子生物學研究所
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摘要: 由D. radiodurans菌體中選殖出一條長約1.2 kb之NAAAR基因,可轉譯出375個胺基酸,其分子量約為40 kDa。此酵素在60℃、pH8.0反應條件下具有最佳之酵素活性。NAAAR活性可明顯被二價金屬離子Co2+與Mn2+所提升。基質選擇性分析顯示,NAAAR具有廣泛之作用基質範圍。NAAAR屬於enolase superfamily中MLE (muconate lactonizing enzyme) subgroup類蛋白質,此類蛋白質的酵素催化反應包含有cycloisomerization (MLE)、dehydration [o-succinylbenzoate synthase (OSBS)]與1,1-proton transfer [L-Ala-D/L-Glu epimerase (AEE)]。本研究探討NAAAR在D. radiodurans菌體中扮演之生理功能。利用Dixon plot分析結果得知,NAAAR之活性易被OSBS之類似物 (salicyl hydroxamate) 所抑制,測得其Ki值為0.68 mM,此結果顯示,salicyl hydroxamate與NAAAR之反應基質作用在酵素相同的位置上,當少量之salicyl hydroxamate存在下,即可與NAAAR的反應基質競爭酵素活性部位。以LC/MS/MS與結合酵素反應分析顯示,NAAAR無法催化L-Ala-D/L-Glu dipeptide進行消旋化反應,也就是不具有AEE酵素活性。NAAAR也無法催化cis,cis-muconate進行cycloisomerization反應,顯示無MLE酵素活性。進一步分析D. radiodurans基因體序列,也無法由資料庫中獲取可提供研判生理功能的相關資訊。因此NAAAR在D. radiodurans中真正的生理功能值得再深入探討。依據NAAAR蛋白質結構分析顯示在酵素活性區域中包含有Lys170、Asp195、Glu220、Asp245與Lys269,其中Asp195、Glu220與Asp245推測與二價金屬離子結合有關,而Lys170與Lys269剛好位於活性部位兩相對位置上,可能負責催化消旋反應的進行。以定點突變方式分析Lys170與Lys269兩個殘基,並測試所有變異酵素之生化活性,結果顯示經突變後所有變異酵素之活性明顯降低約200至1400倍左右,因此,推測Lys170與Lys269在酵素催化消旋反應中扮演非常重要的功能。
The N-acylamino acid racemase (NAAAR) gene from Deinococcus radiodurans BCRC12827 consists of an 1.2 kb open reading frame, encoding a protein of 375-amino acid residues with a calculated molecular mass of about 40 kDa. NAAAR had maximal activity at 60℃and pH 8.0. The high enzyme activity could be observed by the addition of 2mM Co2+ and Mn2+ ion. Substrate specificity analysis revealed that the NAAAR has a broad substrate range. The NAAAR is a member of the MLE (muconate lactonizing enzyme) subgroup of the enolase superfamily, catalyzing the reactions including cycloisomerization (MLE), dehydration [o-succinylbenzoate synthase (OSBS)], and 1,1-proton transfer [L-Ala-D/L-Glu epimerase (AEE)]. Dixon plot analysis showed that NAAAR activity was competitively blocked by the OSBS inhibitor, salicyl hydroxamate, with a Ki of 0.68 mM, indicating that NAc-Met and salicyl hydroxamate bind to the same substrate site of NAAAR. Based on the amino acid sequences identity and protein structure, we proposed that the NAAAR might has AEE function. LC/MS/MS analysis and coupling enzyme assay revealved that NAAAR cannot catalyze the racemization of L-Ala-D/L-Glu. The NAAAR also showed no cycloisomerization activity to cis,cis-muconate. Our data indicated that the physiological function of NAAAR in D. radiodurans is still unclear. Analysis of NAAAR 3-D structure and site-directed mutagenesis implied Lys170 and Lys269 located at opposite side of the active site might be involved in the reversible racemization reaction.
URI: http://hdl.handle.net/11455/21828
其他識別: U0005-1207200710400900
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1207200710400900
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