Structural Studies and 3D Structure Determination of Recombinant <FONT FACE=symb

重组体的结构研究和 3D 结构测定 <FONT FACE=symb

• 批准号: 6433157
• 负责人: Pradman K Qasba
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6433157
• 关键词: Escherichia coli; X ray crystallography; active sites; crystallization; enzyme activity; enzyme structure; galactosyltransferases; inclusion body; lactalbumin; method development; protein structure function; recombinant proteins; site directed mutagenesis; solubility; structural biology

During the FY 1998-1999 we continued the investigations on the structural and functional aspects of the members of beta-1,4-galactosyltransferase enzyme (beta4Gal-T) family, a sub-family of glycosyltransferase super-family that is involved in the synthesis of complex oligosaccharides of glycoconjugates. Each beta4Gal-T family member transfers galactose from UDP-alpha-D-galactose to N acetylglucosamine (GlcNAc), with an ?inversion? of the anomeric configuration at the C1 carbon atom of galactose, generating a beta1-4-linkage. However, each member shows differences in the sugar acceptor specificities and interacts differently with a-lactalbumin (alpha-LA), which modifies the acceptor substrate specificity of the enzyme to glucose. In addition of its transfer of Gal from UDP-alpha-D-galactose to GlcNAc, beta4Gal-T1 also transfers Glc from UDP-alpha-D-glucose to GlcNAc comprising its glucosyltransferase (Glc-T) activity, albeit at an efficiency of 0.3-0.4% of Gal-T activity. We have shown that in the presence of alpha-LA the Glc-T activity of beta4Gal-T1 is enhanced by nearly 30 fold, corresponding to an efficiency of about 10% of the Gal-T activity of the enzyme. By site directed mutagenesis we have identified Trp198 in the recombinant beta4Gal-T1, located within a non-conserved aromatic region, 197YWLY200, to be at least partially involved in binding both the sugar moiety of the sugar-nucleotide donor and the alpha-LA. The apparent K_m of W198A mutant with respect to both UDP-alpha Gal and UDP-alpha-Glc in the GalT- and GlcT-reactions, respectively, decreases compared to the wild type protein. The catalytic turnover number, K_cat, and the catalytic efficiency, K_cat/K_m, both decrease significantly with the mutant protein. However, in the presence of alpha-LA, the apparent K_m for UDP-alpha-Gal and Glc increases, and as well the apparent K_m for alpha-LA increases compared to the wild type, indicating that Trp-198 may be positioned at the interface of the sugar-nucleotide binding site and the site at which beta4Gal-T1 interacts with alpha-LA forcing a conformational change(s) within the enzyme-metal-sugar nucleotide complex in a way that dictates the selection of the acceptor molecule for the reaction. We have also shown that mutation of Cys-342 to Thr increases the in vitro folding efficiency of beta4Gal-T1 from the inclusion bodies by 2 to 3 fold while maintaining the structural integrity and enzymatic activity of the protein. The enzymatic activity has an absolute requirement for Mn+2. Other metal ions, e.g. Co+2, Zn+2, Cd+2, and Fe+2, also activate beta4Gal-T1, albeit to a lesser extent compared to Mn+2. Two metal binding sites, I and II, have been proposed for the beta4-Gal-T1. Site I has an absolute requirement for manganese (K_d = 2 x 10-6 M) and does not bind Ca+2. The second metal binding site can bind Ca+2 and activate the enzyme at low Mn+2 concentrations (10-5 M). Kinetic studies show that the Gal-T activity of the wild type and the mutants of site I, the DXD motif, D244N and D252E, can be activated by Ca+2 in the presence of a low concentration of Mn+2 (2 microM). However, the mutants of site II, E317D, D320N and D320E cannot be activated by Ca+2, even at higher Mn+2 concentrations (20 microM). On the other hand at a fixed Mn+2 concentration (20 microM), Co+2 activates D320N, and D320E to the same level as in the absence of Mn+2, but the wild type GT-d129 is inhibited. In recent years, taking advantage of EST sequences, at least six different family members of beta-4Gal-T, T1 to T6, have been identified in the human genome which exhibit high sequence identity in the catalytic domain of enzyme (80% to 40%). Each member of the family has been shown to be expressed in a tissue specific manner. Among the family members beta4Gal-T4 has only 8% of galactosyltransferase activity compared to that of beta4Gal-T1, the enzyme present in milk. However, in the presence of alpha-LA, beta4Gal-T4 activity increases to nearly 100% of beta-4Gal-T1. This is in contrast to beta4Gal-T1, where alpha-LA enhances the transfer of Gal to glucose rather than to GlcNAc. By site-directed mutational analysis we have identified F280 and F360 in bovine beta4Gal-T1, the residues when mutated to Thr and Met, respectively, that are present at the corresponding positions in beta4Gal-T4, alters beta4Gal-T1 so that it exhibited beta4Gal-T4 property. Thus, it seems that these two Phe mutations may be primarily responsible for the basic characteristics of beta4Gal-T4.We have also cloned and expressed in E. coli the catalytic domain of bovine alpha-1,3-galactosyltransferase, the residues 80-368 of the enzyme, and obtained in soluble form a pure and active protein. The enzyme transfers galactose from UDP-alpha-D-galactose to N-acetylglucosamine (GlcNAc), with the ?retention? of the anomeric configuration at the C1 carbon atom of galactose, generating an alpha1-3-linkage. We have studied the activity of enzyme towards various acceptor substrates, including LacNAc, which is the natural substrate of the enzyme and correlated the activities with the preferred conformation of these substrates derived by molecular dynamics simulations.1) B. Ramakrishnan, P. S Shah, E. Boeggeman and P. K. Qasba. Glycoconjugate J. 16: S74, 19992) E. Boeggeman and P. K. Qasba. Glycobiology 8: Abstract # 141, 1998.

在1998-1999财政年度期间，我们继续研究β-1，4-半乳糖基转移酶（β 4Gal-T）家族成员的结构和功能方面，该家族是糖基转移酶超家族的一个亚家族，参与糖缀合物的复杂寡糖的合成。每个β 4Gal-T家族成员转移半乳糖从UDP-α-D-半乳糖的N乙酰葡萄糖胺（GlcNAc），与？反转？在半乳糖的C1碳原子处的异头构型的，产生β 1 -4-键。然而，每个成员在糖受体特异性方面显示出差异，并且与α-乳白蛋白（α-LA）的相互作用不同，这改变了酶对葡萄糖的受体底物特异性。除了将Gal从UDP-α-D-半乳糖转移到GlcNAc之外，β 4Gal-T1还将Glc从UDP-α-D-葡萄糖转移到GlcNAc，包括其葡糖基转移酶（Glc-T）活性，尽管效率为Gal-T活性的0.3-0.4%。我们已经表明，在α-LA存在下，β 4Gal-T1的Glc-T活性增强了近30倍，相当于酶的Gal-T活性的约10%的效率。通过定点诱变，我们已经鉴定了重组β 4 Gal-T1中位于非保守芳香区197 YWLY 200内的Trp 198，其至少部分参与结合糖-核苷酸供体的糖部分和α-LA。与野生型蛋白相比，W198 A突变体在GalT-和GlcT-反应中分别相对于UDP-α Gal和UDP-α-Glc的表观_Km降低。突变蛋白的催化转换数_Kcat和催化效率_Kcat/_Km均显著降低。然而，在α-LA的存在下，UDP-α-Gal和Glc的表观_Km增加，并且与野生型相比，α-LA的表观_Km也增加，表明Trp-198可以位于糖-核苷酸结合位点和β 4 Gal-T1与α-LA相互作用的位点的界面处，从而以指示选择用于反应的受体分子的方式迫使酶-金属-糖核苷酸复合物内的构象变化。我们还表明，Cys-342突变为Thr使β 4Gal-T1从包涵体的体外折叠效率增加2至3倍，同时保持蛋白质的结构完整性和酶活性。酶活性对Mn+2有绝对要求。其他金属离子，例如Co+2、Zn+2、Cd+2和Fe+2，也激活β 4Gal-T1，尽管与Mn+2相比程度较低。两个金属结合位点，I和II，已被提出的β 4-Gal-T1。位点I对锰有绝对需求（_Kd= 2 x 10-6 M），并且不结合Ca+2。第二个金属结合位点可以结合Ca+2，并在低Mn+2浓度（10-5 M）下激活酶。动力学研究表明，在低浓度Mn+2（2 μ M）存在下，Ca+2可激活野生型和位点I突变体（DXD基序D244 N和D252 E）的Gal-T活性。然而，位点II的突变体E317 D、D320 N和D320 E不能被Ca+2激活，即使在较高的Mn+2浓度（20 μ M）下也是如此。另一方面，在固定的Mn+2浓度（20 μ M）下，Co+2将D320 N和D320 E活化至与不存在Mn+2时相同的水平，但野生型GT-d129被抑制。近年来，利用EST序列，在人类基因组中发现了至少六种不同的β-4Gal-T家族成员（T1至T6），它们在酶的催化结构域中表现出高度序列同一性（80%至40%）。该家族的每个成员已被证明以组织特异性方式表达。在家族成员中，β 4Gal-T4与β 4Gal-T1（存在于牛奶中的酶）相比仅具有8%的半乳糖基转移酶活性。然而，在α-LA的存在下，β-4Gal-T4活性增加到β-4Gal-T1的几乎100%。这与β 4Gal-T1相反，其中α-LA增强Gal向葡萄糖而不是向GlcNAc的转移。通过定点突变分析，我们已经鉴定了牛β 4Gal-T1中的F280和F360，当分别突变为Thr和Met时，存在于β 4Gal-T4中相应位置的残基改变了β 4Gal-T1，使得其表现出β 4Gal-T4特性。因此，这两个Phe突变可能是β_4Gal-T_4基本特性的主要原因。大肠杆菌中的牛α-1，3-半乳糖基转移酶的催化结构域，该酶的残基80-368，并以可溶形式获得纯的活性蛋白。该酶将半乳糖从UDP-α-D-半乳糖转移到N-乙酰葡萄糖胺（GlcNAc），与？保留？在半乳糖的C1碳原子处的异头构型的，产生α 1 -3-键。我们已经研究了酶对各种受体底物的活性，包括LacNAc，其是酶的天然底物，并且通过分子动力学模拟将活性与这些底物的优选构象相关联。1）B. Ramakrishnan，P. S Shah，E. Boeggeman和P.K.卡斯巴。Glycoconjugate J. 16：S74，19992）E. Boeggeman和P.K.卡斯巴。糖生物学8：摘要#141，1998。