Analysis of stereospecificity control mechanism in microbial lactonohydrolase.

微生物内酯水解酶立体特异性控制机制分析。

• 批准号: 09660087
• 负责人: KATAOKA Michihiko
• 金额: $ 2.5万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09660087/
• 关键词: Lactone-ring-cleaving enzyme; stereo specificity; Fusarium oxysporum; Brevibacterium; Lacfonase; Fusarium; 結晶構造解析; パントラクトン; ラクトン; 開裂酵素; Brevibacterium protophormiae; 立体選択的

Elucidation of the reaction and stereospecificity-control mechanisms of microbial stereospecific lactonohydrolases were carried out through structure-function analyses of the enzymes from a fungal strain Fusarium oxysporum and a bacterial strain Brevibacterium protophormiae.As to the enzyme of F.oxysporum, the amino acid sequences of the NH_2 terminus and internal peptide fragments of the enzyme were determined to prepare synthetic oligonucleotides as primers for the PCR.An approximate 1,000-base genomic DNA fragment thus amplified was used as the probe to clone both genomic DNA and cDNA for the enzyme. The lactonohydrolase genomic gene consists of six exons separated by five short introns. The enzyme was crystallized by the vapor-diffusion procedure in the presence of PEG 4,000 as a precipitant. The crystals belong to the monoclinic space group P2_1 with unit-cell parameters a = 156, b = 100, c = 94.1 A, beta = 91.7。.The enzyme of B.protophormiae was isolated and characterized in some detail. The amino acid sequences of NH_2 terminus and internal peptide fragments of the enzyme were determined. On based on these sequence data, synthetic oligonucleotides were prepared as primers for the PCR.Genomic DNA fragment was amplified, and cloning of genomic DNA for the enzyme using this amplified fragment as the probe is now in progress.

通过对一株尖孢镰刀菌（Fusarium oxysporum）和一株原hormiae短杆菌（Brevibacterium protophormiae）的酶的结构-功能分析，阐明了微生物立体特异性乳酸水解酶的反应及其立体特异性调控机制。对尖孢镰刀菌的酶进行了NH_2端和内部肽片段的氨基酸序列测定，制备了人工合成的寡核苷酸作为引物进行PCR。这样扩增的大约1000个碱基的基因组DNA片段被用作探针来克隆该酶的基因组DNA和cDNA。内酯水解酶基因组基因由6个外显子组成，外显子由5个短内含子隔开。酶在PEG 4000作为沉淀剂存在下通过蒸汽扩散程序结晶。晶体属于单斜空间群P2_1，单位胞参数为a = 156, b = 100, c = 94.1 a, β = 91.7。对原乳螨的酶进行了分离和鉴定。测定了酶的NH_2端和内部肽片段的氨基酸序列。在这些序列数据的基础上，制备合成的寡核苷酸作为引物进行PCR。基因组DNA片段被扩增，利用扩增片段作为探针克隆酶的基因组DNA正在进行中。

项目成果

Kataoka, M.et al.: "Crystalliztion and preliminary X-ray diffraction study of lactonohydrolase from Fusarium oxysporum." Acta Cryst.D54. 1432-1434 (1998)

Kataoka, M.et al.：“尖镰孢内酯水解酶的结晶和初步 X 射线衍射研究”。

Kataoka, M.et al.: "Purification and characteriztion of L-allo-threonine aldolase from Aeromonas jandaei DK-39." FEMS Microbiol. Lett.151. 245-248 (1997)

Kataoka, M.et al.：“来自 Aeromonas jandaei DK-39 的 L-别-苏氨酸醛缩酶的纯化和表征。”

Kataoka, M.et al.: "Isolation and characterization of D-threonine aldolase, a pyridoxal-5'-phosphate-dependent enzyme from Arthrobacter sp." Eur.J.Biochem.248. 385-393 (1997)

Kataoka, M.等人：“D-苏氨酸醛缩酶的分离和表征，这是一种来自节杆菌属的吡哆醛-5-磷酸依赖性酶。”

Kataoka, M.et al.: "Enzymatic production of ethyl (R)-4-chloro-3-hydroxybutanoate : asymmetric reduction of ethyl 4-chloro-3-oxobutanoate." Appl.Microbiol.Biotechnol.48. 699-703 (1997)

Kataoka, M.et al.：“(R)-4-氯-3-羟基丁酸乙酯的酶促生产：4-氯-3-氧代丁酸乙酯的不对称还原。”

Kataoka, M.et al.: "Escherichia coli transformant expressing the glucose dehydro-genase gene from Bacillus megaterium as a cofactor regenerator." Biosci.Biotechnol.Biochem.62. 167-169 (1998)

Kataoka, M.等人：“表达来自巨大芽孢杆菌的葡萄糖脱氢酶基因作为辅助因子再生剂的大肠杆菌转化体。”