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標題: Mutational analysis of the active sites of the D-amino acid oxidase from Trigonopsis variabilis
利用定點圖變的方法分析 Trigonopsis variabilis D-型胺基酸氧化脢的 活性位置
作者: 朱曉霞
Ju, Sheau-Shya
關鍵字: 突變;Trigonopsis variabi;氧化脢;Trigonopsis variabilis;D-amini acid oxidase;mutation
出版社: 分子生物研究所
Trigonopsis variabilis 的 D-amino acid oxidase 能催化 D- 型胺基
酸或頭胞菌素 C(cephalosporin C) 進行氧化脫氨作用, 產生 A-型銅酸,
氨及過氧化氫 (H2O2). 在製藥工業上, Dao 脢是產生頭胞菌素類抗生素
之前驅物 7-ACA 的重要酵素, 此酵素屬於flavo-enzyme, 分子量為
39kDa, 需與 FAD 結合才具有活力, 但其活性會受到 H2O2 的抑制. 以定
點突變法將 Dao 脢的胺基酸序列上的 methionine (start codon 除外)
改變成 leu-cine, cysteine 改變成 alanine, 共得到 7 種變異 Dao
脢. 其中, Asp-206-Glu 具有較高的催化效率, 為野生型之 20%, 由於
aspartate 和 glutamate 的測鏈均有 carboxylgroup, 因此推測
Asp-206 為 FAD 的結合位置. Asp-206 可能利用其 carboxyl group與
FAD 以氫鍵結合.
D-amino acid oxidase (Dao, EC1.4.4.4) is an important enzyme for
the bio-synthesis of 7-amino -cephalosporanic acid (7- ACA), the
precursor of cephemantibiotics. The molecular mass of
Trigonopsis variabilis Dao is about 39kDa.This enzyme consists
of two identical subunits and needs FAD for its function.Several
amino acid residues in the conserved region of T. variabilis Dao
were substituted to evaluate the effect of these amino acids on
the enzyme activity. The dao gene was cloned into M13mp19 for
site-directed mutagenesis. DNA frag-ments containing the mutated
dao gene were subcloned into expression vector pOE30 to
facilitate the recovery of the mutated Dao enzymes. Change of
His-324with Glu, Tyr, Ala, Asn and Gln retained about 30~90% of
Dao activity and the Km of the mutated Dao His-324-Ala, His-324-
Asn and His-324-Gln was increased. These results indicated that
His-324 might be involved in substrate binding.Replacement of
Asp-206 with Leu, Gly, and Asn losed the characteristic absorp-
tion spectrum of flavoenzyme and abolished the Dao activity
completely. Sub-stitution of Asp-206 with Glu, Ala, and Ser
retained about 30~90% Dao activity.Dao mutants of Asp-206-Glu,
Asp-206-Ala, and Asp-206-Ser exhibited similar spectral profile
as wild-type Dao enzyme did. The results suggested that Asp-206
might play an important role in the binding of FAD to Dao.
Appears in Collections:分子生物學研究所

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