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

在1998-1999年度，我们继续对β -1,4-半乳糖转移酶（β - 4gal - t）家族成员的结构和功能方面的研究，β -1,4-半乳糖转移酶是糖基转移酶超家族的一个亚家族，参与糖缀合物的复杂低聚糖的合成。每个beta4Gal-T家族成员都能将半乳糖从udp - α - d -半乳糖转化为N -乙酰氨基葡萄糖（GlcNAc），并发生反转。在半乳糖的C1碳原子上产生一个- 1-4键。然而，每个成员在糖受体特异性上表现出差异，与a-乳清蛋白（α -la）的相互作用也不同，后者改变了该酶对葡萄糖的受体底物特异性。除了将葡萄糖从udp - α - d -半乳糖转移到GlcNAc之外，beta4Gal-T1还将葡萄糖从udp - α - d -葡萄糖转移到GlcNAc，包括其葡萄糖基转移酶（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α - la存在下，beta4Gal-T1的Glc-T活性提高了近30倍，相当于酶的Gal-T活性提高了约10%。通过定点诱变，我们在重组蛋白beta4Gal-T1中发现Trp198位于一个非保守的芳香区197YWLY200中，至少部分参与了糖核苷酸供体和α - la的糖段结合。与野生型蛋白相比，W198A突变体在GalT-和glct -反应中与udp - α - Gal和udp - α - glc相关的表观K_m减小。催化转化率K_cat，催化效率K_cat/K_m随突变蛋白的增加而显著降低。然而，在α - la存在的情况下，与野生型相比，udp - α - gal和Glc的表观K_m增加，α - la的表观K_m增加。这表明，Trp-198可能位于糖-核苷酸结合位点和β - 4gal - t1与α - la相互作用位点的界面，迫使酶-金属-糖核苷酸复合物内的构象发生变化，从而决定了反应的受体分子选择。我们还发现，将Cys-342突变为Thr可使beta4Gal-T1在包涵体上的体外折叠效率提高2 -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，如Co+2、Zn+2、Cd+2和Fe+2，也能激活beta4Gal-T1，尽管与Mn+2相比作用较小。两个金属结合位点，I和II，已被提出用于β - gal - t1。Site I对锰有绝对需求（K_d = 2 × 10-6 M），并且不结合Ca+2。第二个金属结合位点可以在低Mn+2浓度（10-5 M）下结合Ca+2并激活酶。动力学研究表明，在低浓度Mn+2（2微米）存在下，野生型和1位点突变体DXD基元D244N和D252E的Gal-T活性可以被Ca+2激活。然而，位点II、E317D、D320N和D320E的突变体即使在较高的Mn+2浓度（20微米）下也不能被Ca+2激活。另一方面，在固定Mn+2浓度（20微米）下，Co+2对D320N和D320E的激活作用与不含Mn+2时相同，但对野生型GT-d129的抑制作用较弱。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，从T1到T6，在酶的催化结构域具有较高的序列同源性（80% ~ 40%）。该家族的每个成员都以特定的组织方式表达。在家族成员中，β - 4gal - t4的半乳糖转移酶活性仅为β - 4gal - t1的8%，这种酶存在于牛奶中。然而，在α - la的存在下，β - 4gal - t4的活性几乎增加到β - 4gal - t1的100%。这与β - 4gal - t1相反，α - la增强Gal向葡萄糖的转移，而不是向GlcNAc转移。通过位点定向突变分析，我们在牛β - 4gal - t1中鉴定出F280和F360，分别在β - 4gal - t4中对应位置突变为Thr和Met的残基改变了β - 4gal - t1，使其表现出β - 4gal - t4的特性。因此，这两个Phe突变可能是beta4Gal-T4基本特征的主要原因。我们还克隆并在大肠杆菌中表达了牛α -1,3-半乳糖基转移酶的催化结构域，酶的残基80-368，并以可溶性形式获得了纯活性蛋白。该酶将半乳糖从udp - α - d -半乳糖转化为n -乙酰氨基葡萄糖（GlcNAc），并保留？在半乳糖的C1碳原子上产生一个α 1-3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，并通过分子动力学模拟将活性与这些底物的首选构象联系起来。1) B. Ramakrishnan， P. S . Shah， E. Boeggeman， P. K. Qasba。杨建军，张建军。糖苷与糖苷的结合[j] .糖苷与糖苷学报，1999,11(2):1 - 4。糖苷生物学8：摘要# 141,1998。

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.