Evidence for essential catalytic determinants for human erythrocyte pyrimidine 5′-nucleotidase

Human erythrocyte pyrimidine 5′-nucleotidase, PN-I, catalyzes the dephosphorylation of pyrimidine nucleoside monophosphates. The enzyme also possesses phosphotransferase activity, transferring phosphate groups between pyrimidine nucleoside monophosphates and various pyrimidine nucleosides. Deficiency of the enzyme activity is associated with a hemolytic anemia. PN-I cDNA has been expressed in Escherichia coli, yielding a fully active recombinant enzyme, which was purified to homogeneity and extensively characterized. Multiple sequence alignment of PN-I and homologues proteins revealed the existence of conserved regions, whose importance in catalysis was examined by performing experiments designed to intercept covalent intermediates as strongly suggested by our previous kinetic studies. Furthermore, a functional analysis of the enzyme was carried out through site-directed mutagenesis designed on the basis of the sequence of the identified conserved regions as well as mutations observed in PN-I-deficient patients.Received 25 March 2005; received after revision 3 May 2005; accepted 13 May 2005     

丝裂霉素诱变黑曲霉产植酸酶过程模型化

用均匀设计分析方案,进行丝裂霉素C对黑曲霉化学诱变菌丝体和原生质体诱导产植酸酶选育,并以诱变时间(X_1)和丝裂霉素C的浓度(X_2)建立回归方程．实验筛选出具有4株较高酶活的菌株,诱变菌丝体得到2株酶活是始发菌株183．89％和147．55％;诱变原生质体得到2株酶活是始发菌株395．52％和381． 98％．由响应面模型初步判断,在丝裂霉素C浓度为5～30 mg·L-1和诱变时间45～60 min时,丝裂霉素C 对黑曲霉诱变原生质体选育产植酸酶菌株的效果比对菌丝体诱变效果好．用均匀设计对丝裂霉素C诱变菌丝体和原生质体的效果进行回归分析,所得模型调整后的相关系数分别为89．02％和99．13％,模型拟合程度好,实验误差小,可以用此模型较好的对丝裂霉素C的黑曲霉菌丝体诱变进行分析和预测．     

Co-transformation to tobacco of Cre/lox site-specific recombination system and its precise recombination

For the temporally and spatially regulated expression of the barnase gene in plant, two kinds of plasmids with cre gene and its directly repeat recognition sitesiox from bacteriophage P1 were constructed and co-transformed into tobacco by agrobacterium mediated procedure. The transgenic plants were conformed by PCR analysis. The blocking fragment between the twolox directly repeat sites was excised by Cre protein in the transgenic plant genome. Cloning and sequencing the DNA fragment from the co-transformed plant DNA showed that the precise DNA excision occurred in transgenic tobacco genome directed by Cre/lox site-specific recombination.     

一株耐高温L-乳酸高产菌的选育及其发酵特征

对鼠李糖乳杆菌菌株Lactobacillus rhamnosus JCM1553进行紫外诱变,选育得到一株耐高温L-乳酸高产突变菌GX-6.在47℃,分别以葡萄糖和木薯淀粉为底物,研究GX-6菌株发酵生产L-乳酸的情况并考察GX-6菌株的遗传稳定性.结果表明,以葡萄糖为底物时,发酵56h的L-乳酸产量达到117g/L,...     

细胞色素P450 配体通道的研究进展

细胞色素P450 (CYP)是一类含亚铁血红素的酶, 不但能催化内源性底物的生物合成和代谢, 而且对外源性化合物的代谢、激活以及降解毒性等起着重要作用. P450 也是人体内最主要的药物代谢酶, 能催化代谢约75%的临床药物. 已有的P450 酶晶体结构显示, 绝大多数酶呈现闭合的构象, 其催化活性位点位于血红素上方且深埋于蛋白质的中心, 没有明显的通道用于配体进出活性中心. 因此一个有趣而重要的问题是, 配体如何进出酶的活性中心达到被氧化或发生抑制作用? 近年来, 关于P450 酶配体通道的研究取得了显著进展. 本文重点综述了通道研究的实验方法及6 类P450 酶可能存在的通道和作用机制, 并对其未来的发展方向进行了展望.     

A链链内二硫键移位突变胰岛素原的构建与表达

采用定点突变技术,将人胰岛素原基因编码框中A11位氨基酸残基与A12位氨基酸残基进行了互换,构建了[CysA11Ser,SerA12Cys]人胰岛素原突变基因.在大肠杆菌原核表达系统中对突变体基因进行了表达,并对表达产物进行了变复性研究及初步的分离纯化,获得了具有稳定结构的突变体蛋白质,探讨了突变体A链链内二硫键的形成状况,为进一步深入揭示A链链内二硫键的角色与功能打下了基础.     

饲用菜籽饼微生物脱毒研究

从霉菌（Ｍｏｕｌｄ）、酵母菌（Ｙｅａｓｔ）、乳酸细菌（Ｌａｃｔｉｃｂａｃｔｅｒｉａ）中选育出对菜籽饼粕中的抗营养因子具有强降解能力的根霉（Ｒｈｉｚｏｐｕｓ ｓｐｐ．）、白地霉（Ｇｅｏｔｒｉｃｈｕｍ ｃａｎｄｉｄｕｍ）、酿酒酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）、乳酸杆菌（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｓｐｐ．）。采用纯培养、混种固态发酵法对菜籽粕进行微生物脱毒试验，初步研究结果表明     

DS 9701菌株的紫外诱变及PHB解聚酶高产菌株的筛选

以青霉(Penicillium.sp)DS 9701为出发菌株,通过紫外线诱变分生孢子,采用透明圈初筛和摇瓶培养复筛的方法,获得一突变株,编号DS 9701-M 09.该突变株的PHB解聚酶活力是原始菌株的2.3倍,经传代培养,该菌株的PHB解聚酶高产特性稳定遗传.     

蛋白质分子的定点突变和化学修饰

回顾蛋白质工程研究及应用技术、手段和方法,分析定点突变及化学修饰在改造蛋白质分子结构与功能方面的异同。定点突变技术可以随心所欲地在已知DNA序列中取代、插入或缺失一定长度的核苷酸片段。该方法与使用化学因素导致突变的方法相比,具有突变率高、简单易行、重复性好的特点。     

Site-directed mutagenesis reveals new and essential elements for iron-coordination of the sulfur oxygenase reductase from the acidothermophilic Acidianus tengchongensis

Previous study on refolding of sulfur oxygenase reductase (SOR) inclusion bodies from recombinant Escherichia coli showed that iron was critical to the activity of the SOR from Acidianus ambivalens. In this study, enzymatic assays showed that 2,2′-Dipyridyl, Tiron and 8-hydroxyquinoline, which are specific for chelating ferrous or ferric ions, strongly inhibited the activity of SOR from A. tengchongensis, suggesting that iron atom is essential for SOR activity. Alignment of several functionally identified SORs and SOR-like sequences from genome database revealed a conserved, putative iron binding motif, H⁸⁶-X₃-H⁹⁰-X _n -E¹¹⁴-X _n -E¹²⁹ (numbering according to the Acidianus tengchongensis SOR sequence). Three mutants of SOR were generated by site-directed mutagenesis of H₈₆, H₉₀ and E₁₂₉ into phenylalanine or alanine residue in this study. Circular dichroism spectrum determination indicated that there was no change of the secondary structures of mutant SORs, H⁸⁶F, H⁹⁰F and E¹²⁹A, but all mutants were completely inactive. Through determination of iron contents we found that SOR mutants of H⁸⁶F, H⁹⁰F and E¹²⁹A completely or partially lost iron, while mutants of C³¹S, C¹⁰¹S, and C¹⁰⁴S (generated in a previous study) did not. This result indicated that H⁸⁶, H⁹⁰ and E¹²⁹ but not C³¹, C¹⁰¹, and C¹⁰⁴ were involved in binding to iron atom. Based on this and previous studies, it is proposed that the conserved motifs, C³¹-X _n -C¹⁰¹-X₂-C¹⁰⁴ and H⁸⁶-X₃-H⁹⁰-X₂₃-E¹¹⁴-X₁₄-(E/D)¹²⁹, are respectively for sulfur and molecular oxygen binding and activation. These two conserved motifs are essential elements for the SOR activity. Supported by National Natural Science Foundation of China (Grant Nos. 30670018, 30621005) and State Key Basic Research and Development Program of China (Grant No. 2004CB719602)