Structure and Function of Enzymes Participating in Metabolism of D-Amino Acid of Bacterial Cell Walls and Development of their Specific Inhibitors
Structure and Function of Enzymes Participating in Metabolism of D-Amino Acid of Bacterial Cell Walls and Development of their Specific Inhibitors
批准号:
06454077
负责人:
SODA Kenji
金额:
$5.06万
依托单位:
依托单位国家:
日本
项目类别:
Grant-in-Aid for General Scientific Research (B)
财政年份:
1994
资助国家:
日本
项目状态:
已结题
起止时间:
1994 至 1995
中文摘要
D-氨基酸如D-丙氨酸和D-谷氨酸是细菌细胞壁的肽聚糖层的不可缺少的组分。因此,参与D-氨基酸生物合成的细菌酶的特异性抑制剂可以是有效的抗生素。本论文对丙氨酸消旋酶、D-氨基酸氨基转移酶和谷氨酸消旋酶的作用机制进行了详细的研究,旨在开发基于机制的酶抑制剂。我们研究了嗜热脂肪芽孢杆菌的热稳定丙氨酸消旋酶的Lys 39的作用,该酶与辅因子吡哆醛5 '-磷酸(PLP)结合,并通过定点突变提出该残基是催化碱基。Lys 39的末端氨基作为碱从底物中提取2-氢。烷基胺对K39 A突变酶的影响表明,Lys 39作为唯一的催化碱从底物中提取2-氢,并将其返回到去质子化中间体的底物部分的2-碳。 关于我们 吃过了D-氨基酸氨基转移酶催化各种D-氨基酸和酮酸之间的氨基转移。我们研究了热稳定D-氨基酸氨基转移酶的亮氨酸201残基的催化作用:该残基在晶体学上显示在活性位点附近,通过定点突变与结合的PLP相互作用。Leu 201残基可能在PMP转化为PLP的过程中调节辅因子的功能。谷氨酸消旋酶催化谷氨酸消旋生成D-谷氨酸。我们比较了这些酶与乳酸菌。加入UDP-N-乙酰胞壁酰-L-丙氨酸(大肠杆菌酶的激活剂)影响大肠杆菌酶的CD和荧光光谱。相反,戊糖原杆菌和短乳杆菌的酶不被UDP-N-乙酰胞壁酰-L-丙氨酸激活。片球菌酶显示出与哺乳动物肌红蛋白的显著序列相似性,并且被氯化血红素抑制。在不存在UDP-N-乙酰胞壁酰-L-丙氨酸的情况下,氯化血红素不抑制大肠杆菌的谷氨酸消旋酶,但在存在UDP-N-乙酰胞壁酰-L-丙氨酸的情况下强烈抑制谷氨酸消旋酶。通过添加UDP-N-乙酰胞壁酰-L-丙氨酸,大肠杆菌酶的构象被转化为与片球菌酶的构象相似的形式。少
英文摘要
D-Amino acids such as D-alanine and D-glutamate are the indespensable components of the peptidoglycan layr of the bacterial cell walls. Thus, the specific inhibitors for the bacteral enzymes participate in the biosyntheses of D-amino acids can be potent antibiotics. We studied detailed mechanisms of alanine racemase, D-amino acid aminotransferase, and glutamate racemase, to develop the meachanism-based inhibitors for these enzymes. We studied the role of Lys39 of the thermostable alanine racemase of Bacillus stearothermophilus, which is bound to the cofactor, pyridoxal 5'-phosphate (PLP) , and suggested this residue to be a catalytic base by site-directed mutagenesis. The terminal amino group of Lys39 acts as a base to abstract the 2-hydrogen from the substrate. The effects of alkylamines on the K39A mutant enzymes suggest that Lys39 acts as a sole catalytic base to abstract the 2-hydrogen from the substrate and returns it to the 2-carbon of substrate moiety of a deprotonated intermedi … More ate. D-Amino acid aminotransferase catalyzes the transfer of amino group between various D-amino acids and keto acids. We studied the catalytic role of leucine 201 residue of the themostable D-amino acid aminotransferase : the residue was crystallographically shown to be in the vicinity of the active-site to interact with the bound PLP by site-directed mutagenesis. The Leu 201 residue probably regulates the function of cofactor during the conversion of PMP to PLP.Glutamate racemase catalyzes the racemization of glutamate to produce D-glutamate. We compared the enzyme with those of Lactic acid bacteria. The addition of UDP-N-acetylmuramyl-L-alanine, an activator of the E.coli enzyme affected the CD and fluorescence spectra of the E.coli enzyme. In contrast, the enzymes of P.pentosaceus and L.brevis were not activated by UDP-N-acetylmuramyl-L-alanine. The Pediococcus enzyme shows a significant sequence similarity to mammalian myoglobin, and was inhibited by hemin. Glutamate racemase of E.coli was not inhibited by hemin in the absence of UDP-N-acetylmuramyl-L-alanine, but strongly inhibited in the presence of UDP-N-acetylmuramyl-L-alanine. The conformation of the E.coli enzyme is converted to a similar form to that of the Pediococcus enzyme by the addition of UDP-N-acetylmuramyl-L-alanine. Less
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K.Kishimoto et al.: "Role of Leucine 201 of Thermostable D-Amino Acid Aminotrasferase from a Thermophile." J.Biochem.(印刷中). (1994)
K.Kishimoto 等人:“来自嗜热菌的热稳定 D-氨基酸氨基转移酶的亮氨酸 201 的作用”。J.Biochem(出版中)。
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通讯作者:
Ashiuchi Makoto: "In Vivo Effect of GroESL on the Folding of Glutamate Racemase of Eschirichia coli." J.Biochem.117. 495-498 (1995)
Ashiuchi Makoto:“GroESL 对大肠杆菌谷氨酸消旋酶折叠的体内影响。”
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H. Toyama et al.: "Reconstitute of Fragmentary Form of Thermostable Alanine Racemase" Biosci. Biotech. Biochem.59. 1118-1120 (1995)
H. Toyama 等人:“热稳定丙氨酸消旋酶片段形式的重构”Biosci。
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Nishimura Katsushi: "A Simple Method for Detemination of Stereospecificity of Aminotransferase for C-4′Hydrogen Transfer of the Coenzyme." Bioorganic & Medical Chemistry. 2. 605-607 (1994)
Katsushi Nishimura:“测定辅酶 C-4 氢转移的氨基转移酶立体特异性的简单方法。”2. 605-607 (1994)
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Choi, Soo-Young: "Bacterial Glutamate Racemase has High Sequence Homology with Myoglobins and Forms an Equimolar Inactive Complex with Hemin." Proc.Natl.Acad.Sci., USA. 91. 10144-10147 (1994)
Choi, Soo-Young:“细菌谷氨酸消旋酶与肌红蛋白具有高序列同源性,并与氯化血红素形成等摩尔的非活性复合物。”
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共 17 条
Epidemiologic and behavioral survey of HIV/AIDS in Cambodia
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批准号:09041189
-
项目类别:Grant-in-Aid for international Scientific Research
-
资助金额:$2.5万
-
财政年份:1997
-
负责人:SODA Kenji
-
依托单位:
Structural and functional analysis of bacterial D-amino acid metabolic enzymes to develop their inhibitors
-
批准号:08456052
-
项目类别:Grant-in-Aid for Scientific Research (B)
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资助金额:$5.12万
-
财政年份:1996
-
负责人:SODA Kenji
-
依托单位:
Epidemiologic survey of actual situation of HIV/AIDS in Cambodia
-
批准号:07041165
-
项目类别:Grant-in-Aid for international Scientific Research
-
资助金额:$6.27万
-
财政年份:1995
-
负责人:SODA Kenji
-
依托单位:
Biochemical functions and metabolisms of D-amino acid
-
批准号:07308048
-
项目类别:Grant-in-Aid for Scientific Research (A)
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资助金额:$12.1万
-
财政年份:1995
-
负责人:SODA Kenji
-
依托单位:
STUDIES ON THE USEFULNESS OF SALIVA TO DETECT ANTIBODIES AGAINST HIV IN EPIDEMIOLOGICAL RESEACH
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批准号:06454241
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项目类别:Grant-in-Aid for General Scientific Research (B)
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资助金额:$3.33万
-
财政年份:1994
-
负责人:SODA Kenji
-
依托单位:
Protein engineering studies of thermostable D-amino acid transaminase
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批准号:05044095
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项目类别:Grant-in-Aid for international Scientific Research
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资助金额:$3.84万
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财政年份:1993
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负责人:SODA Kenji
-
依托单位:
Follow-up studies on protective effects of breast feeding against rotavirus infection in infants.
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批准号:04670335
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项目类别:Grant-in-Aid for General Scientific Research (C)
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资助金额:$0.96万
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财政年份:1992
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负责人:SODA Kenji
-
依托单位:
Studies on Protective Effects of Breast Feeding Against Rotavirus Infection in Infants.
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批准号:02670244
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项目类别:Grant-in-Aid for General Scientific Research (C)
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资助金额:$1.15万
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财政年份:1990
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负责人:SODA Kenji
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依托单位:
Design of New Selenium Enzymes and Proteins, and Their Functions
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批准号:02454545
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项目类别:Grant-in-Aid for General Scientific Research (B)
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资助金额:$4.03万
-
财政年份:1990
-
负责人:SODA Kenji
-
依托单位:
Development of New Methods on Isotope-Labeling of Amino Acid and Vitamin with Microbial Enzymes
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批准号:63880021
-
项目类别:Grant-in-Aid for Developmental Scientific Research (B).
-
资助金额:$5.12万
-
财政年份:1988
-
负责人:SODA Kenji
-
依托单位:
Sero-epidemiological Study of Herpes Simplex Virus
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批准号:60570259
-
项目类别:Grant-in-Aid for General Scientific Research (C)
-
资助金额:$1.22万
-
财政年份:1985
-
负责人:SODA Kenji
-
依托单位:
Enzymatic Synthesis of Optically-active Selenium and Tellurium Containing Amino Acids and the Reaction Mechanism
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批准号:60560113
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项目类别:Grant-in-Aid for General Scientific Research (C)
-
资助金额:$1.15万
-
财政年份:1985
-
负责人:SODA Kenji
-
依托单位:
海外基金