大肠杆菌O152中wfgD基因编码的β-1,3-葡萄糖基转移酶产物结构的质谱表征

大肠杆菌O152抗原的寡糖重复单位中含葡萄糖-β-1,3-N-乙酰葡萄糖胺(Glc-β-1,3-GlсNAc)连接键。本研究采用电喷雾离子化多级串联质谱技术对以人工合成的天然受体底物的结构类似物苯氧基十一烷二磷酸-N-乙酰葡萄糖胺(GlcNAc-β-PO3-PO3-(CH2)11-O-phenyl(GlcNAc-PP-PhU))为受体底物,尿苷二磷酸葡萄糖(UDP-Glc)为给予体底物的酶促反应产物进行了详细的结构表征。电喷雾离子化多级串联质谱图中观察到的主要碎片源于磷酸二酯键部分和糖苷键的裂解。此外,由观察到的二糖产物非还原端碎片获得了序列信息;跨环断裂碎片及源于吡喃环取代基消除的‘内在’碎裂离子可提供组成产物的单糖残基连接方式信息。广泛的碎裂信息表明wfgD基因编码UDP-Glc:GlcNAc-pyrophosphate-lipid中的β-1-3葡萄糖基转移酶。     

基质辅助激光解吸电离-飞行时间质谱法检测罕见单糖合成酶

以VioF(普罗威登斯菌O30中O抗原基因簇内)编码的甲酰基转移酶酶促反应产物为研究对象,采用碰撞诱导解离(CID)负离子模式基质辅助激光解吸电离-飞行时间质谱(MALDI-TOF MS)技术建立简单、高效的罕见单糖合成酶监测方法。本方法首先直接质谱分析0.3μL未经色谱分离、除盐处理的酶促反应混合物,随后应用CID串联质谱技术对酶活反应产物进行结构表征,实现酶活反应的快速监测。结果表明:高能CID MALDI-TOF/TOF-MS平台适用于新建克隆的单糖合成酶酶活反应的监测,与现有的监测方法相比,在速度、灵敏度、重现性、自动化和溶剂消耗方面具有绝对优势。     

Globo H四糖衍生物的高效合成

设计合成了2个Globo H四糖衍生物1和2, 将其作为标准样品可用于研究β1,3-葡萄糖醛酸(GlcA)转移酶及GlcA-3-O-硫酸化(Sulfo)转移酶在肿瘤组织内的特异性表达.     

HIV-1嵌合抗原的纯化及免疫原性分析

为获得高效的HIV诊断试剂,选定HIV-1的外膜蛋白env中469-511aa,538-674aa和700-734aa3处包含较多抗原位点的区域作为免疫抗原,用PCR的方法从HIV-1全基因扩增编码这3处片段的基因序列,将它们克隆到原核表达载体中,利用大肠杆菌表达系统表达嵌合蛋白.结果发现,3段嵌合基因能在大肠杆菌BL21(Star)中表达,通过Ni-sepharose4B金属Ni螯合层析柱分离纯化目的产物,酶联免疫检测结果表明,纯化抗原有较强的抗原特异性.     

基于二氧化硅荧光纳米颗粒与核酸染料SYBRGreenⅠ的双色显微成像技术用于E.coliO157:H7的检测

将免疫荧光纳米标记技术与激光共聚焦显微成像方法相结合,发展了一种基于二氧化硅荧光纳米颗粒和核酸染料SYBR Green Ⅰ的双色显微成像技术用于大肠杆菌O157:H7的检测.采用联吡啶钌(RuBpy)二氧化硅荧光纳米颗粒对羊抗大肠杆菌O157:H7抗体进行修饰,基于抗体-抗原相互作用实现了其对目标大肠杆菌O157:H7...     

Rif-orf13编码的细胞色素P450催化利福霉素生物合成过程中C34a位的羟化反应（英文）

利福霉素生物合成途径在经历了二十余年的研究之后,仍然没有得到完全阐明.其中C34a甲基的氧化脱除是利福霉素成熟过程中的必需反应步骤,但是催化这一步骤的酶尚未鉴定;推测可能是利福霉素生物合成基因簇编码的某个细胞色素P450催化了这一步骤.选取利福霉素生物合成基因簇中功能尚未确证的P450基因rif-orf0、rif-orf4和rif-orf13在变铅青链霉菌中进行异源表达和底物喂养实验,发现表达了rif-orf13的链霉菌能够将16-脱甲基-34a-脱氧利福霉素W (1)转化为16-脱甲基利福霉素W (2).将rif-orf13在大肠杆菌BL21 (DE3)中进行诱导表达,利用纯化的Orf13蛋白进行体外酶催化反应,发现Orf13能够将底物1羟化为产物2.结合前人的基因敲除研究,认为rif-orf13是编码34a-脱氧利福霉素W羟化酶的基因,其在胞内的功能可以被另一个负责C12-C29双键氧化断裂的P450基因rif-orf5替代.     

基于二氧化硅荧光纳米颗粒与核酸染料SYBRGreenⅠ的双色显微成像技术用于E.coliO157:H7的检测

将免疫荧光纳米标记技术与激光共聚焦显微成像方法相结合,发展了一种基于二氧化硅荧光纳米颗粒和核酸染料SYBR Green Ⅰ的双色显微成像技术用于大肠杆菌O157:H7的检测.采用联吡啶钌(RuBpy)二氧化硅荧光纳米颗粒对羊抗大肠杆菌O157:H7抗体进行修饰,基于抗体-抗原相互作用实现了其对目标大肠杆菌O157:H7的特异性标记;同时以核酸染料SYBR Green Ⅰ对细菌进行染色,将细菌和纳米颗粒团聚体区分开,实现了对大肠杆菌O157:H7的双色标记,并通过激光共聚焦显微镜进行免分离的荧光成像检测.结果表明,该方法可用于缓冲溶液体系和混合细菌样品中目标大肠杆菌O157:H7的特异性检测,在仅含5％目标菌的混合样品中仍能观察到具有明显黄色荧光的大肠杆菌O157:H7,且整个检测步骤包括样品预处理可在3h内完成.该方法则具有较好的灵敏度,可检出2.6×103 Cell/mL的目标细菌样品.若采用针对其它病原菌细胞壁抗原的特异性抗体,则有望发展成为一种通用技术用于多种病原菌的快速和灵敏检测.     

以草酸及咪唑基配体构筑的二维网状锌(Ⅱ)配位聚合物的合成、晶体结构及荧光性质

通过水热法合成了一个新的配位聚合物[Zn(C2O4)(1,3-bix)]n(1,H2C2O4=草酸,1,3-bix=1,3-双(咪唑基-1-甲基)-苯)。并对其进行了元素分析、红外光谱、热重和X-射线单晶衍射测定。该配合物属于三斜晶系,P1空间群。在晶体结构中,锌原子为六配位与来自2个不同的C2O42-配体上的4个羧基氧原子和来自2个不同的1,3-bix配体上的2个氮原子配位,呈现畸变的八面体几何构型。而且该配合物通过氢键和π-π堆积作用扩展成三维超分子网状结构。此外还研究了它的荧光性质。     

两个由双咪唑基配体构筑的锰、镉配合物的合成及晶体结构(英文)

通过水热法合成了2个金属-有机配位聚合物[Mn(dipha)(1,3-bix)]n·nH2O(1)和[Cd2(NIPH)2(bimb)2.5(H2O)]n·3nH2O(2)(H2dipha=2,2′-联苯二甲酸,1,3-bix=1,3-双(咪唑基-1-甲基)-苯,H2NIPH=5-硝基间苯二甲酸,bimb=1,4-双(咪唑基-1-甲基)-丁烷)。并对其进行了元素分析、红外光谱、热重和X-射线单晶衍射测定。这2个配合物通过氢键和π-π相互作用形成了超分子网状结构。此外还研究了配合物2的荧光性质。     

1,2和1,3-缩水内醚糖衍生物的制备, 构象及其糖苷化反应

吡喃糖的1,2-及1,3-缩水内醚苄醚由相应的吡喃糖的C-2氧负离子(对1,3-缩水内醚是C-3氧负离子)与连有氯原子的C-1经分子内关环反应而制备, 而有些吡喃糖的1,2-缩水内醚苄醚是由相应的吡喃糖的C-1氧负离子与连有对甲苯磺酰氧基的C-2经分子内关环(倒关环)反应而得。呋喃糖的1,2-缩水内醚苄醚只能用倒关环法合成。1,2-(或1,3-)缩水内醚糖的开环反应通常给出1,2-反式连接(对1,3缩水内醚是1,3反式连接)的糖苷键。1,2-及1,3-缩水内醚糖的构象分析是通过^1H NMR测定及分子力学计算的方法而完成的。     

A Native Ternary Complex Trapped in a Crystal Reveals the Catalytic Mechanism of a Retaining Glycosyltransferase

Glycosyltransferases (GTs) comprise a prominent family of enzymes that play critical roles in a variety of cellular processes, including cell signaling, cell development, and host–pathogen interactions. Glycosyl transfer can proceed with either inversion or retention of the anomeric configuration with respect to the reaction substrates and products. The elucidation of the catalytic mechanism of retaining GTs remains a major challenge. A native ternary complex of a GT in a productive mode for catalysis is reported, that of the retaining glucosyl‐3‐phosphoglycerate synthase GpgS from M. tuberculosis in the presence of the sugar donor UDP‐Glc, the acceptor substrate phosphoglycerate, and the divalent cation cofactor. Through a combination of structural, chemical, enzymatic, molecular dynamics, and quantum‐mechanics/molecular‐mechanics (QM/MM) calculations, the catalytic mechanism was unraveled, thereby providing a strong experimental support for a front–side substrate‐assisted S_Ni‐type reaction.     

Discovery of new biocatalysts for the glycosylation of terpenoid scaffolds

The synthesis of terpenoid glycosides typically uses a chemical strategy since few biocatalysts have been identified that recognise these scaffolds. In this study, a platform of 107 recombinant glycosyltransferases (GTs), comprising the multigene family of small molecule GTs of Arabidopsis thaliana have been screened against a range of model terpenoid acceptors to identify those enzymes with high activity. Twenty-seven GTs are shown to glycosylate a diversity of mono-, sesqui- and diterpenes, such as geraniol, perillyl alcohol, artemisinic acid and retinoic acid. Certain enzymes showing substantial sequence similarity recognise terpenoids containing a primary alcohol, irrespective of the linear or cyclical structure of the scaffold; other GTs glycosylate scaffolds containing secondary and tertiary alcohols; the carboxyl group of other terpenoids also represents a feature that is recognized by GTs previously known to form glucose esters with many different compounds. These data underpin the rapid prediction of potential biocatalysts from GT sequence information. To explore the potential of GTs as biocatalysts, their use for the production of terpenoid glycosides was investigated by using a microbial-based whole-cell biotransformation system capable of regenerating the cofactor, UDP-glucose. A high cell density fermentation system was shown to produce several hundred milligrams of a model terpenoid, geranyl-glucoside. The activities of the GTs are discussed in relation to their substrate recognition and their utility in biotransformations as a complement or alternative to chemical synthesis.     

Enzymatic biosynthesis of benzylisoquinoline alkaloid glycosides via promiscuous glycosyltransferases from Carthamus tinctorius

Three new GTs (UGT84A33, UGT71AE1 and UGT90A14) from Carthamus tinctorius exhibited robust catalytic promiscuity to benzylisoquinoline alkaloids, and were used as enzymatic tools for the synthesis of diverse benzylisoquinoline alkaloid glycosides.     

Biochemical and structural insights of the early glycosylation steps in calicheamicin biosynthesis

The enediyne antibiotic calicheamicin (CLM) gamma(1)(I) is a prominent antitumor agent that is targeted to DNA by a novel aryltetrasaccharide comprised of an aromatic unit and four unusual carbohydrates. Herein we report the heterologous expression and the biochemical characterization of the two "internal" glycosyltransferases CalG3 and CalG2 and the structural elucidation of an enediyne glycosyltransferase (CalG3). In conjunction with the previous characterization of the "external" CLM GTs CalG1 and CalG4, this study completes the functional assignment of all four CLM GTs, extends the utility of enediyne GT-catalyzed reaction reversibility, and presents conclusive evidence of a sequential glycosylation pathway in CLM biosynthesis. This work also reveals the common GT-B structural fold can now be extended to include enediyne GTs.     

Insights into the synthesis of lipopolysaccharide and antibiotics through the structures of two retaining glycosyltransferases from family GT4

Glycosyltransferases (GTs) catalyze the synthesis of the myriad glycoconjugates that are central to life. One of the largest families is GT4, which contains several enzymes of therapeutic significance, exemplified by WaaG and AviGT4. WaaG catalyses a key step in lipopolysaccharide synthesis, while AviGT4, produced by Streptomyces viridochromogenes, contributes to the synthesis of the antibiotic avilamycin A. Here we present the crystal structure of both WaaG and AviGT4. The two enzymes contain two "Rossmann-like" (beta/alpha/beta) domains characteristic of the GT-B fold. Both recognition of the donor substrate and the catalytic machinery is similar to other retaining GTs that display the GT-B fold. Structural information is discussed with respect to the evolution of GTs and the therapeutic significance of the two enzymes.     

Enzymatic glucosylation of unnatural naphthols by a promiscuous glycosyltransferase from Aloe arborescens

Enzymatic glucosylation of unnatural products by natural glycosyltransferases (GTs) has great potential in creating novel and bioactive glucosides. A new GT (AaGT3) from Aloe arborescens exhibited catalytic promiscuity and high efficiency to diverse unnatural naphthols. By combing the substrate flexibility and catalytic reversibility of AaGT3, a cost-effective enzymatic approach to novel and bioactive unnatural glucosides was established. These studies indicate the significant potential of promiscuous natural GTs in synthesis of unnatural bioactive glucosides in drug discovery.     

Structural Snapshots of α‐1,3‐Galactosyltransferase with Native Substrates: Insight into the Catalytic Mechanism of Retaining Glycosyltransferases

Glycosyltransferases (GTs) are a key family of enzymes that catalyze the synthesis of glycosidic bonds in all living organisms. The reaction involves the transfer of a glycosyl moiety and can proceed with retention or inversion of the anomeric configuration. To date, the catalytic mechanism of retaining GTs is a topic of great controversy, particularly for those enzymes containing a putative nucleophilic residue in the active site, for which the occurrence of a double‐displacement mechanism has been suggested. We report native ternary complexes of the retaining glycosyltransferase α‐1,3‐galactosyltransferase (α3GalT) from Bos taurus , which contains such a nucleophile in the active site, in a productive mode for catalysis in the presence of its sugar donor UDP‐Gal, the acceptor substrate lactose, and the divalent cation cofactor. This new experimental evidence supports the occurrence of a front‐side substrate‐assisted S_Ni‐type reaction for α3GalT, and suggests a conserved common catalytic mechanism among retaining GTs.     

Two sequence elements of glycosyltransferases involved in urdamycin biosynthesis are responsible for substrate specificity and enzymatic activity.

BACKGROUND: Two deoxysugar glycosyltransferases (GTs), UrdGT1b and UrdGT1c, involved in urdamycin biosynthesis share 91% identical amino acids. However, the two GTs show different specificities for both nucleotide sugar and acceptor substrate. Generally, it is proposed that GTs are two-domain proteins with a nucleotide binding domain and an acceptor substrate site with the catalytic center in an interface cleft between these domains. Our work aimed at finding out the region responsible for determination of substrate specificities of these two urdamycin GTs. RESULTS: A series of 10 chimeric GT genes were constructed consisting of differently sized and positioned portions of urdGT1b and urdGT1c. Gene expression experiments in host strains Streptomyces fradiae Ax and XTC show that nine of 10 chimeric GTs are still functional, with either UrdGT1b- or UrdGT1c-like activity. A 31 amino acid region (aa 52-82) located close to the N-terminus of these enzymes, which differs in 18 residues, was identified to control both sugar donor and acceptor substrate specificity. Only one chimeric gene product of the 10 was not functional. Targeted stepwise alterations of glycine 226 (G226R, G226S, G226SR) were made to reintroduce residues conserved among streptomycete GTs. Alterations G226S and G226R restored a weak activity, whereas G226SR showed an activity comparable with other functional chimeras. CONCLUSIONS: A nucleotide sugar binding motif is present in the C-terminal moiety of UrdGT1b and UrdGT1c from S. fradiae. We could demonstrate that it is an N-terminal section that determines specificity for the nucleotide sugar and also the acceptor substrate. This finding directs the way towards engineering this class of streptomycete enzymes for antibiotic derivatization applications. Amino acids 226 and 227, located outside the putative substrate binding site, might be part of a larger protein structure, perhaps a solvent channel to the catalytic center. Therefore, they could play a role in substrate accessibility to it.     

Retaining glycosyltransferase mechanism studied by QM/MM methods: lipopolysaccharyl-α-1,4-galactosyltransferase C transfers α-galactose via an oxocarbenium ion-like transition state

Glycosyltransferases (GTs) catalyze the highly specific biosynthesis of glycosidic bonds and, as such, are important both as drug targets and for biotechnological purposes. Despite their broad interest, fundamental questions about their reaction mechanism remain to be answered, especially for those GTs that transfer the sugar with net retention of the configuration at the anomeric carbon (retaining glycosyltransferases, ret-GTs). In the present work, we focus on the reaction catalyzed by lipopolysaccharyl-α-1,4-galactosyltransferase C (LgtC) from Neisseria meningitides. We study and compare the different proposed mechanisms (S(N)i, S(N)i-like, and double displacement mechanism via a covalent glycosyl-enzyme intermediate, CGE) by using density functional theory (DFT) and quantum mechanics/molecular mechanics (QM/MM) calculations on the full enzyme. We characterize a dissociative single-displacement (S(N)i) mechanism consistent with the experimental data, in which the acceptor substrate attacks on the side of the UDP leaving group that acts as a catalytic base. We identify several key interactions that help this front-side attack by stabilizing the transition state. Among them, Gln189, the putative nucleophile in a double displacement mechanism, is shown to favor the charge development at the anomeric center by about 2 kcal/mol, compatible with experimental mutagenesis data. We predict that using 3-deoxylactose as acceptor would result in a reduction of k(cat) to 0.6-3% of that for the unmodified substrates. The reactions of the Q189A and Q189E mutants have also been investigated. For Q189E, there is a change in mechanism since a CGE can be formed which, however, is not able to evolve to products. The current findings are discussed in the light of the available experimental data and compared with those for other ret-GTs.     

Bismuth determination in environmental samples by hydride generation-electrothermal atomic absorption spectrometry

A hydride generation procedure, via flow injection, coupled to electrothermal atomic absorption spectrometry was optimised for Bi determination in sea water and hot-spring water and acid extracts from coal, coal fly ash and slag samples. The effects of several variables such as hydrochloric acid and sodium tetrahydroborate concentrations, hydrochloric acid and sodium tetrahydroborate flow rates, reaction coil length, trapping and atomisation temperatures, trapping time and the Ar flow rate have been investigated by using a 2⁹*3/128 Plackett-Burman design. From these studies, certain variables (sodium tetrahydroborate concentration and trapping time) showed up as significant, and they were optimised by a 2²+star central composite design. In addition, a study of the bismuthine trapping and atomisation efficiency from graphite tubes (GTs) permanently treated with uranium, tantalum, lanthanum oxide, niobium, beryllium oxide, chromium oxide and tantalum carbide were investigated. The results obtained were compared with those achieved by iridium and zirconium-treated GTs. The best analytical performances, with characteristic mass of 35 pg and detection limit of 70 ng l⁻¹, were achieved by using U-treated GTs. Accuracy were checked using several reference materials: 1643d (Trace Elements in Water), TM-24 (Reference Water), GBW-07401 (Soil) and 1632c (Trace Elements in Coal).