天然产物的糖基化及糖基侧链改造

抗生素和抗癌药物等多种天然产物的活性都依赖于其糖基侧链,糖基侧链结构的变化对母体化合物的生物活性、底物适应性及药理学性质具有重要影响。糖基侧链结构变化多端,修饰、改变天然产物的糖基侧链已成为获得临床候选药物的重要方法。利用化学法和酶法,研究者创造了多种改造天然产物糖基化的方法。详细介绍了天然产物的糖基化过程,并从组合生物学、糖基转移酶改造、糖类随机化及新型糖类随机化和糖基转移酶可逆性四方面阐述了糖基侧链的改造方法。     

微生物合成黄酮糖苷类天然产物研究进展

黄酮糖苷类天然产物是植物中黄酮类化合物的主要存在形式,通过糖基化修饰,可以改变其水溶性、稳定性等,赋予其新的生物活性和功能。黄酮类化合物的糖基化修饰通常由植物源或微生物源的糖基转移酶催化,根据糖基的位置、类型和数量的不同,可形成多种类型的黄酮糖苷类产物。随着合成生物学和代谢工程的快速发展,在微生物中合成植物源黄酮糖苷类天然产物取得了重要进展。综述了糖基转移酶的聚类分析及糖基供体的途径改造,并对代谢工程优化黄酮糖苷类天然产物的微生物合成进行了分析讨论,并对其发展前景进行了展望。     

天然产物的C-糖基化研究进展

自然界中的天然产物在其结构上存在各种修饰,C-糖基化为其中一种比较稀少的修饰方式.C-C糖苷键的形成由C-糖基转移酶负责催化.含C-糖基的化合物多数来自微生物,但高等植物也会有少量积累.综述了近些年来在天然产物C-糖基化方面的研究工作,并对其在药物开发方面的潜力进行了展望.     

植物源UDP-糖基转移酶及其分子改造*

糖基化能够增加化合物的结构多样性,有效改善水溶性、药理活性和生物利用度,对植物天然产物的药物开发至关重要。UDP-糖基转移酶(UGTs)能够催化糖基从活化的核苷酸糖供体转移到受体形成糖苷键,植物中天然产物的糖基化修饰主要通过UGTs实现。但是大多数天然UGTs的催化活性、稳定性和底物特异性较低,难以满足工业用酶的要求,限制了它们的工业化应用。近年来,通过分子改造技术改进天然UGTs的催化特性取得了突破性的研究进展。为此,概述了植物源UGTs的挖掘与表征、三维结构和催化机制,归纳了UGTs分子改造的思路和方法,包括理性设计和定向进化,重点介绍了结构域替换、序列保守分析和结构分析与定点突变的结合,总结了定向进化中的高通量筛选方法,为植物天然产物酶法糖基化的工业应用提供了参考和借鉴。     

植物糖基转移酶的结构与机理及糖基化工程的研究进展

糖基转移酶(glycosyltransferases, GTs)广泛存在于各种有机体中,通过糖基化反应参与维持细胞代谢稳态.糖基转移酶能够识别多种受体,催化活化的糖基从供体分子转移到受体分子上,改变受体分子的化学稳定性、水溶性以及受体分子的转运能力和生物活性等,进而有助于提高其生物利用度和生物活性等.许多被糖基化修饰的化合物成为药物分子的重要来源.然而,天然产物中的糖苷类化合物存在含量低、提取难度大和提取产物纯度差等问题.在利用化学合成方法合成糖苷类化合物的过程中,无法实现特定位点的糖基化修饰,同时原料试剂和副产物易对环境造成污染.因此,近年来对糖基转移酶的研究日渐增多.本文简要综述了植物糖基转移酶的结构和生物技术应用的研究进展,为基于植物糖基转移酶结构的糖基化工程和生物活性糖苷化合物的生产提供有用信息.     

天然产物糖基转移酶的分类、进化及底物预测研究进展

糖基化是一种十分重要且独特的天然产物结构修饰方式,该修饰由天然产物糖基转移酶(natural product glycosyltransferases, NP-GTs)催化完成,可以改变底物的理化性质或生物学活性。本文首先从结构、催化机制与糖基化位点的角度总结了NP-GTs的分类;其次,从生物来源和底物类别两个角度分析了NPGTs的进化关系;介绍了NP-GTs底物预测的三种常用策略;最后,展望了NP-GTs的研究趋势,为开展基于糖基化修饰的先导化合物优化、蛋白质设计、合成生物学等研究提供参考。     

植物激素糖基化修饰研究进展

植物激素对植物的生长发育有重要的调节作用。由于激素的作用依赖于其浓度, 所以植物内源活性激素的水平必须受到严格控制, 而糖基化修饰被认为是调控激素活性水平的重要方式之一。随着植物激素糖基化修饰相关糖基转移酶基因不断被克隆与鉴定, 多种植物激素的糖基化修饰机制和功能作用逐渐被揭示。该文重点介绍了近年来植物生长素、细胞分裂素、脱落酸、油菜素内酯、水杨酸、茉莉酸等植物激素的糖基转移酶活性鉴定与功能研究进展。同时, 对植物激素糖基化修饰领域存在的问题和发展前景进行了讨论。     

酵母N-糖基化工程研究进展

酵母表达系统可用来生产具生物活性的重组糖蛋白,但其在N-糖基化过程中会生成高甘露糖型糖链。通过引入相关的甘露糖苷酶和糖基转移酶基因、切断酵母自身的高甘露糖链形成通道能够改变酵母宿主N-糖基化的类型。本对酵母N-糖基化工程的研究状况、最新进展及存在问题作简要阐述。     

常见致病菌糖基转移酶的结构与功能

糖基转移酶(glycosyltransferases,GTs)将糖基从活化的供体转移到糖、脂、蛋白质和核酸等受体,其参与的蛋白质糖基化是最重要的翻译后修饰(post-translational modifications,PTMs)之一。近年来越来越多的研究证明,糖基转移酶与致病菌毒力密切相关,在致病菌的黏附、免疫逃逸和定殖等生物学过程中发挥关键作用。目前,已鉴定的糖基转移酶根据其蛋白质三维结构特征分为3种类型GT-A、GT-B和GT-C,其中常见的是GT-A和GT-B型。在致病菌中发挥黏附功能的糖基转移酶,在结构上属于GT-B或GT-C型,对致病菌表面蛋白质(黏附蛋白、自转运蛋白等)进行糖基化修饰,在致病菌黏附、生物被膜的形成和毒力机制发挥具有重要作用。糖基转移酶不仅参与致病菌黏附这一感染初始过程,其中属于GT-A型的一类致病菌糖基转移酶会进入宿主细胞,通过糖基化宿主蛋白质影响宿主信号传导、蛋白翻译和免疫应答等生物学功能。本文就常见致病菌糖基转移酶的结构及其糖基化在致病机制中的作用进行综述,着重介绍了特异性糖基化高分子量(high-molecular-weight,HMW)黏附蛋白的糖基转移酶、针对富丝氨酸重复蛋白(serine-rich repeat proteins,SRRP)糖基化修饰的糖基转移酶、细菌自转运蛋白庚糖基转移酶(bacterial autotransporter heptosyltransferase,BAHT)家族、N-糖基化蛋白质系统和进入宿主细胞发挥毒力作用的大型梭菌细胞毒素、军团菌(Legionella)葡萄糖基转移酶以及肠杆菌科的效应子NleB。为揭示致病菌中糖基转移酶致病机制的系统性研究提供参考,为未来致病菌的诊断、药物设计研发以及疫苗开发等提供科学依据和思路。     

植物糖基转移酶在植物抗病过程中的研究进展

植物在复杂的环境中进化出了各种反应来应对危害,其中糖基化作用就是植物利用的一种主要的生理机制.糖基化作用通过改变受体化合物的生物活性及其细胞内的定位来降低外物质对自身的影响,从而达到植物体生理代谢的稳态.植物中的糖基转移酶就是专门负责实现这种糖基化反应的酶类.简要概述了糖基转移酶在植物抗性过程中的研究方法、分类及生物学功能,并对其研究方向加以展望.     

Features and applications of bacterial glycosyltransferases: current state and prospects

The bioactivity of many natural products including valuable antibiotics and anticancer therapeutics depends on their sugar moieties. Changes in the structures of these sugars can deeply influence the biological activity, specificity and pharmacological properties of the parent compounds. The chemical synthesis of such sugar ligands is exceedingly difficult to carry out and therefore impractical to establish on a large scale. Therefore, glycosyltransferases are essential tools for chemoenzymatic and in vivo approaches for the development of complex glycosylated natural products. In the last 10 years, several examples of successful alteration and diversification of natural product glycosylation patterns via metabolic pathway engineering and enzymatic glycodiversification have been described. Due to the relaxed substrate specificity of many sugar biosynthetic enzymes and glycosyltransferases involved in natural product biosynthesis, it is possible to obtain novel glycosylated compounds using different methods. In this review, we would like to provide an overview of recent advances in diversification of the glycosylated natural products and glycosyltransferase engineering.     

Achievements and impacts of glycosylation reactions involved in natural product biosynthesis in prokaryotes

Bioactive natural products, such as polyketides, flavonoids, glycopeptides, and aminoglycosides, have been used as therapeutic agents. Many of them contain structurally diverse sugar moieties attached to the aglycone core structures. Glycosyltransferases (GTs) catalyze the attachment of nucleotide-activated sugar substrates to acceptor aglycones. Because these sugar moieties are usually essential for biological activity, in vivo pathway engineering in prokaryotic hosts and in vitro enzymatic approaches coupled with GT engineering are currently being used to synthesize novel glycosylated derivatives, and some of them exhibited improved biological activities compared to the parent molecules. Therefore, harnessing the potential of diverse glycosylation reactions in prokaryotes will increase the structural diversity of natural products and the possibility to generate new bioactive products.     

Engineering the glycosylation of natural products in actinomycetes

Bioactive natural products are frequently glycosylated with saccharide chains of different length, in which the sugars contribute to specific interactions with the biological target. Combinatorial biosynthesis approaches are being used in antibiotic-producing actinomycetes to generate derivatives with novel sugars in their architecture. Recent advances in this area indicate that glycosyltransferases involved in the biosynthesis of natural products have substrate flexibility regarding the sugar donor but also, less frequently, with respect to the aglycon acceptor. Therefore, the possibility exists of altering the glycosylation pattern of natural products, thus enabling an increase in the structural diversity of natural products.     

微藻中的高附加值天然产物与挖掘策略

微藻是一类单细胞的光合自养真核生物,因为生长速度快,油脂含量高,对土地和水资源的要求宽松而被认为是下一代的液体燃料来源。此外,微藻还能够生产多种多样的天然化合物,包括微藻多糖、长链不饱和脂肪酸、色素和生物碱等。与植物中丰富的生物碱研究相比,对微藻中生物碱的研究仍处于起步阶段。微藻中的许多天然产物,通常具有多样性的生物活性,可以作为食品添加剂、营养保健品乃至医药,具有较高的经济价值。本文将简要介绍微藻产生的几类高附加值产品,并就微藻中高附加值天然产物的挖掘策略与规模化培养做简要的探讨。     

Crystal structures of a multifunctional triterpene/flavonoid glycosyltransferase from Medicago truncatula

Glycosylation is a ubiquitous reaction controlling the bioactivity and storage of plant natural products. Glycosylation of small molecules is catalyzed by a superfamily of glycosyltransferases (GTs) in most plant species studied to date. We present crystal structures of the UDP flavonoid/triterpene GT UGT71G1 from Medicago truncatula bound to UDP or UDP-glucose. The structures reveal the key residues involved in the recognition of donor substrate and, by comparison with other GT structures, suggest His-22 as the catalytic base and Asp-121 as a key residue that may assist deprotonation of the acceptor by forming an electron transfer chain with the catalytic base. Mutagenesis confirmed the roles of these key residues in donor substrate binding and enzyme activity. Our results provide an initial structural basis for understanding the complex substrate specificity and regiospecificity underlying the glycosylation of plant natural products and other small molecules. This information will direct future attempts to engineer bioactive compounds in crop plants to improve plant, animal, and human health and to facilitate the rational design of GTs to improve the storage and stability of novel engineered bioactive compounds.     

微生物来源天然产物中的β-甲基色氨酸单元及其生物合成研究进展

微生物在次级代谢过程中通常会产生结构复杂、活性多样的天然产物。这些天然产物是新药发展的基础,亦可作为先导化合物或重要的药效基团用于药物研发。结构多样的氨基酸单元是参与合成复杂多样天然产物的重要前体。天然产物中的β-甲基氨基酸单元不仅可以赋予其生物活性,还能增强其生物稳定性而不被肽酶水解。本文综述了含有β-甲基氨基酸单元的天然产物,尤其对含有β-甲基色氨酸单元的天然产物生物合成途径进行了阐释。对β-甲基色氨酸单元生物合成途径的理解结合基因组数据有助于进行新结构天然产物的挖掘,并为运用代谢科学理念和合成生物学技术开发含有该单元的新化合物提供理论基础和可操作遗传元件。     

Recent advances in glycan synthesis

Carbohydrates play important roles in life science, but their synthesis is always hampered by their complicated chemical structures. Scientists have never stopped trying to solve the problem of glycan synthesis from various aspects. Here a brief overview of recent progress in glycan synthesis, including chemical approaches, chemoenzymatic approaches, and automated synthesis, will be discussed, focusing on the efficiency of new glycosylation methods, the stereoselectivity of coupled products, and their applications in the assembly of complex glycan chains.     

Evaluation of human N-linked glycosylation sites in murine granulocyte-macrophage colony-stimulating factor.

Nonglycosylated murine and human granulocyte-macrophage colony-stimulating factor have a molecular mass of approximately 14.5 kDa predicted from the primary amino acid sequence. The expression of both proteins in COS cells leads to a heterogeneous population of molecules that differ in the degree of glycosylation. Both human and murine molecules contain two N-linked glycosylation sites that are situated in nonhomologous locations along the linear sequence. Despite this difference both proteins show a similar size distribution among the glycosylation variants. These studies analyze the effects of introducing in the murine protein novel N-linked glycosylation sites corresponding to those sites found in the human molecule. A panel of molecules composed of various combinations of human N-linked glycosylation sites in either the presence or the absence of murine N-linked glycosylation was compared. Substitution of a proper human N-linked glycosylation consensus sequence at Asn 24 did not result in N-linked glycosylation, nor was there any considerable effect on bioactivity. Replacement of the N-linked glycosylation consensus sequence at Asn 34 results in glycosylation similar to that found in the human molecule and causes a significant decrease in bioactivity. These data suggest that the position of N-linked glycosylation is critical for maximal bioactivity in a particular species and that the changes in position of these sites in different species probably occurred during evolution in response to changes in their receptors.     

Requirement of hydrophilic amino-terminal residues for granulocyte-macrophage colony-stimulating factor bioactivity and receptor binding.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein required for the proliferation and differentiation of granulocyte and macrophage precursors. Previous investigations have identified regions in human and murine GM-CSF that are required for bioactivity. In the present study, alanine substitution mutagenesis was undertaken to define more precisely specific amino-terminal residues in murine GM-CSF that are involved in bioactivity and receptor binding. Five double alanine mutants were identified that showed at least 10-fold reductions in bioactivity (K14AK20A, K14AE21A, H15AK20A, H15AE21A, K20AE21A). Each of these mutants maintained a normal N-linked glycosylation pattern when expressed in COS-1 cells, suggesting that native polypeptide backbone conformation was preserved. The purified prokaryotic expression products of two mutants (K14AE21A and H15AE21A) had a 100-fold decrease in bioactivity and a decrease in receptor binding, indicating that the side chains of K14, H15, and E21 are required for optimal receptor binding and maximal bioactivity.