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.

1998-1999财年期间，我们继续研究β-1,4-半乳糖基转移酶（β4Gal-T）家族成员的结构和功能方面，β-1,4-半乳糖基转移酶家族是糖基转移酶超家族的一个亚家族，参与复合糖低聚糖的合成。每个 beta4Gal-T 家族成员都会通过“反转”将半乳糖从 UDP-α-D-半乳糖转移到 N 乙酰葡糖胺 (GlcNAc)。半乳糖 C1 碳原子上的异头构型发生改变，产生 β1-4-连接。然而，每个成员在糖受体特异性方面表现出差异，并且与α-乳清蛋白（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，beta4Gal-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%。我们已经证明，在存在 α-LA 的情况下，β4Gal-T1 的 Glc-T 活性增强了近 30 倍，相当于该酶的 Gal-T 活性的效率约为 10%。通过定点诱变，我们已经鉴定出重组β4Gal-T1中的Trp198，位于非保守芳香区197YWLY200内，至少部分参与糖-核苷酸供体的​​糖部分和α-LA的结合。与野生型蛋白质相比，W198A突变体在GalT-和GlcT-反应中分别相对于UDP-αGal和UDP-α-Glc的表观K m 降低。突变蛋白的催化周转数 K<sub​​>cat</sub> 和催化效率 K<sub​​>cat</sub>/K<sub​​>m</sub> 均显着降低。然而，在存在α-LA的情况下，与野生型相比，UDP-α-Gal和Glc的表观K<sub​​>m</sub>增加，并且α-LA的表观K<sub​​>m</sub>也增加，表明Trp-198可能位于糖核苷酸结合位点和β4Gal-T1与α-LA作用力相互作用的位点的界面处。 酶-金属-糖核苷酸复合物内的构象变化决定了反应受体分子的选择。我们还表明，Cys-342 突变为 Thr 可使包涵体中 β4Gal-T1 的体外折叠效率提高 2 至 3 倍，同时保持蛋白质的结构完整性和酶活性。酶的活性对Mn+2有绝对的要求。其他金属离子，例如Co+2、Zn+2、Cd+2 和 Fe+2 也会激活 beta4Gal-T1，尽管与 Mn+2 相比程度较小。已为 beta4-Gal-T1 提出了两个金属结合位点 I 和 II。位点 I 对锰有绝对需求 (K<sub​​>d</sub> = 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，野生型和位点 I 突变体、DXD 基序、D244N 和 D252E 的 Gal-T 活性可以在低浓度 Mn+2 (2 µM) 存在的情况下被 Ca+2 激活。然而，位点 II、E317D、D320N 和 D320E 的突变体不能被 Ca+2 激活，即使在较高的 Mn+2 浓度 (20 µM) 下也是如此。另一方面，在固定的 Mn+2 浓度（20 µM）下，Co+2 将 D320N 和 D320E 激活至与不存在 Mn+2 时相同的水平，但野生型 GT-d129 受到抑制。近年来，利用EST序列，在人类基因组中鉴定出至少6个不同的β-4Gal-T家族成员（T1至T6），它们在酶的催化结构域中表现出较高的序列同一性（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。在该家族成员中，与牛奶中存在的酶 beta4Gal-T1 相比，β4Gal-T4 的半乳糖基转移酶活性仅为 8%。然而，在存在 α-LA 的情况下，β4Gal-T4 的活性几乎是 β-4Gal-T1 的 100%。这与 beta4Gal-T1 相反，其中 α-LA 增强 Gal 向葡萄糖的转移，而不是向 GlcNAc 的转移。通过定点突变分析，我们在牛 beta4Gal-T1 中鉴定出了 F280 和 F360，当这些残基分别突变为 Thr 和 Met 时（存在于 beta4Gal-T4 中的相应位置），会改变 beta4Gal-T1，使其表现出 beta4Gal-T4 特性。因此，看来这两个Phe突变可能是β4Gal-T4的基本特征的主要原因。我们还在大肠杆菌中克隆并表达了牛α-1,3-半乳糖基转移酶的催化结构域，即该酶的80-368位残基，并以可溶形式获得了纯的活性蛋白。该酶将半乳糖从 UDP-α-D-半乳糖转移至 N-乙酰葡糖胺 (GlcNAc)，并保留“保留”。半乳糖 C1 碳原子上的异头构型发生改变，产生 α1-3-键。我们研究了酶对各种受体底物的活性，包括酶的天然底物 LacNAc，并将活性与通过分子动力学模拟得出的这些底物的优选构象相关联。1) B. Ramakrishnan、P. S Shah、E. Boeggeman 和 P. K. Qasba。糖缀合物 J. 16：S74，19992) E. Boeggeman 和 P. K. Qasba。糖生物学 8：摘要#141，1998。