唾液酸苷酶的催化机理及水解与合成寡糖进展

唾液酸苷酶(EC.3.2.1.18)是一类重要的糖苷水解酶,在动物和微生物中广泛存在.该类酶催化寡糖或糖缀合物上非还原末端唾液酸水解,具有重要的生物学功能,如参与溶酶体降解代谢物、癌症发生、微生物致病等多种生理和病理过程.除了水解活性外,有的唾液酸苷酶还具有转糖基活性,能够以唾液酸单糖或糖苷为糖基供体,催化唾液酸转移到受体分子上,一步合成寡糖和糖苷化合物.这种合成活性对于唾液酸相关糖链的大量获得具有重要意义,有利于推动该类寡糖的基础研究及其在食品和医药中的应用.本文综述了唾液酸苷酶的结构和催化机理、生理功能、转糖基作用及其在寡糖合成中的应用.     

蛇肌醛缩酶和兔肌醛缩酶在构象上的差别以及亚基的相互作用

(1)用硫酸铵分级,然后磷酸纤维素亲和层析得到了均一的蛇肌醛缩酶,凝胶过滤法测定全酶分子量为160,000,SDS-凝胶电泳法测定了它的亚基分子量为40,000,表明蛇肌醛缩酶是一个四亚基的酶,它属于醛缩酶Ⅰ-A 型。(2)甲醇或尿素对蛇肌醛缩酶和兔肌醛缩酶的影响是不同的。当用胰蛋白酶分别消化蛇肌醛缩酶和兔肌醛缩酶时,发现蛇肌醛缩酶对胰蛋白酶的作用更为敏感。甲醇或尿素对酶活性影响的结果说明蛇肌醛缩酶有更紧密的构象,酶分子内部的疏水键在维持活化型时起着重要的作用,在低有机溶剂浓度时不受影响,而在高有机溶剂浓度时分子内部疏水键才遭到破坏。此外蛇肌醛缩酶分子表面的极性基团,包括赖氨酸和精氨酸形成的离子对可能更易受到胰蛋白酶作用,同时它们可阻止低浓度甲醇与酶分子内疏水键作用。(3)醛缩酶和TNBS 之间的反应支持酶分子的氨基,尤其是蛇肌醛缩酶的氨基在维护其天然构象中起着特殊作用的观点。(4)虽然蛇肌醛缩酶和兔肌醛缩酶的构象有一定的不同,但是仍然能够进行杂交产生蛇肌-兔肌醛缩酶杂交株组,说明它们亚基之间的结合区域的差别并不很大。     

蛇肌醛缩酶和兔肌醛缩酶在构象上的差别以及亚基的相互作用

(1)用硫酸铵分级,然后磷酸纤维素亲和层析得到了均一的蛇肌醛缩酶,凝胶过滤法测定全酶分子量为160,000,SDS-凝胶电泳法测定了它的亚基分子量为40,000,表明蛇肌醛缩酶是一个四亚基的酶,它属于醛缩酶I-A型。(2)甲醇或尿素对蛇肌醛缩酶和兔肌醛缩酶的影响是不同的。当用胰蛋白酶分别消化蛇肌醛缩酶和兔肌醛缩酶时,发现蛇肌醛缩酶对胰蛋白酶的作用更为敏感。甲醇或尿素对酶活性影响的结果说明蛇肌醛缩酶有更紧密的构象,酶分子内部的疏水键在维持活化型时起着重要的作用,在低有机溶剂浓度时不受影响,而在高有机溶剂浓度时分子内部疏水键才遭到破坏。此外蛇肌醛缩酶分子表面的极性基团,包括赖氨酸和精氨酸形成的离子对可能更易受到胰蛋白酶作用,同时它们可阻止低浓度甲醇与酶分子内疏水键作用。(3)醛缩酶和TNBS之间的反应支持酶分子的氨基,尤其是蛇肌醛缩酶的氨基在维护其天然构象中起着特殊作用的观点。(4)虽然蛇肌醛缩酶和兔肌醛缩酶的构象有一定的不同,但是仍然能够进行杂交产生蛇肌-兔肌醛缩酶杂交株组,说明它们亚基之间的结合区域的差别并不很大。     

苏氨酸醛缩酶的催化机理、分子改造及合成应用

苏氨酸醛缩酶催化醛和甘氨酸羟醛缩合,一步反应即可构建产物β-羟基-α-氨基酸的两个手性中心,从原子经济性和环境影响角度,是非常具有潜力的绿色合成光学纯β-羟基-α-氨基酸的方式之一。多个不同生物来源的苏氨酸醛缩酶得到分离和表征,较低的β-碳立体选择性以及反应过程中动力学和热力学控制难题,使其在合成应用中面临很大挑战。文中综述了近年来苏氨酸醛缩酶在基因挖掘、催化机理、高通量筛选与分子改造、合成应用等方面的研究进展,旨在为进一步研究提供参考。     

合成唾液酸乳糖重组大肠杆菌的构建

唾液酸寡糖具有抗感染、提高免疫力、促进双歧杆菌增殖等功能,对其微生物合成方法的研究具有重要的理论和应用价值。克隆和表达来源于Campylobacter jejuni的N-乙酰葡萄糖胺异构酶基因(neuC)、乙酰神经氨酸合成酶基因(neuB)、CMP-乙酰神经氨酸合成酶基因(neuA)和来源于Nesseria meningitidis的唾液酸转移酶基因(nst),利用表达载体pSTV29,在大肠杆菌Escherichia coli JM109内构建合成唾液酸乳糖的代谢途径。在接种量2%、装液量50 mL/250 mL三角瓶、摇床转速180 r/min、温度34℃的条件下发酵30 h,并以10 g/L乳糖为底物,利用HPLC检测到唾液酸乳糖的合成量为2.45 g/L,为人乳唾液酸寡糖及其类似物的异体合成提供了新的思路。     

苏氨酸醛缩酶的结构与功能及其在药物合成中的应用

β-羟基-α-氨基酸(β-hydroxy-α-amnio acids,HAAs)是一类广泛应用于制药工业的重要手性中间体。由于其含有双手性中心(C_α和C_β),探索其严格立体选择性的生物合成方法备受关注。苏氨酸醛缩酶(threonine aldolase,TA)可在温和条件下催化不同类型的醛与氨基酸缩合构筑丰富的HAAs产物库,显示了工业应用潜力。由于目前表征的TA普遍存在对C_β立体选择性不严格、活性较低以及催化机制不清晰等问题,为其在HAAs合成中的应用带来了挑战。本文综述了TA在新酶挖掘、结构与催化机理解析、蛋白质工程以及合成应用等方面的研究进展,为推动酶催化绿色、高效合成手性药物提供参考。     

聚唾液酸与唾液酸的研究进展

唾液酸是一族神经氨酸(Neuraminic acid)的衍生物。聚唾液酸(Polysialic acid)是唾液酸(Sialic acid)单体以α-2,8或α-2,9键连接的直链同聚物,是一些哺乳动物细胞中糖蛋白的组成部分和少数几种细菌的胞外多糖组分。综述了唾液酸和聚唾液酸的结构、性质、生物学功能、生物合成和生产应用。     

多聚唾液酸转移酶研究进展

多聚唾液酸(polysialic acid,PSA)是一类线性、均一多聚α2,8连接唾液酸的独特碳水化合物,它主要通过典型的N-连接糖苷键附着在脊椎动物神经系统神经黏附分子(neural cell adhesion molecule,NCAM)上。PSA通过改变NCAM的黏附性调节神经细胞发育、神经导向以及突触形成,从而在神经发育中起关键作用。PSA表达的调节具有时间和结构依赖性,NCAM的唾液酸化是由两种多聚唾液酸转移酶(polysialyltrans ferases)-ST8Sia II(STX)和ST8Sia IV(PST)所催化,它们都属于6个基因编码的α2,8唾液酸转移酶家族。STX和PST都可将多个唾液酸残基转移到含有NeuNAcα2-3(或6)Galβ1-4GlcNAcβ1-R结构的N糖链受体上;两种酶共同作用时远远大于一种酶形成的PSA量。总之,多聚唾液酸转移酶可调节PSA的合成并参与了脊椎动物的神经发育。     

聚唾液酸,一种非GAGs、非免疫原性生物材料的应用研究进展

聚唾液酸是一种由N-乙酰神经氨酸连接、电负性的线性同聚物,存在于人体、动物细胞和少数致病菌中,主要以糖蛋白(神经细胞粘附分子)和糖脂形式存在,是一种非糖胺聚糖(GAGs)、非免疫原性、生物可降解的优良生物材料。聚唾液酸可用作组织工程和药物缓释材料,也可以与其它大分子复合形成功能材料。对聚唾液酸生物学功能、发酵生产及应用进行概述,以期为聚唾液酸的进一步应用研究提供参考。     

两种新型L-苏氨酸醛缩酶的鉴定及活性检测方法

为了实现重要医药中间体p-羟基-α-氨基酸的生物酶法合成,挖掘验证新型的L-苏氨酸醛缩酶.以pET-28a(+)作为表达载体,通过蛋白表达纯化、薄层层析色谱(TLC)和高效液相色谱(HPLC)技术分析L-苏氨酸醛缩酶及其催化产物的性质.基于4-氨基-3-肼基-5-巯基-1,2,4-三氮唑(Purpald)显色试剂开发检...     

Polysaccharide lyases

Abstract: Polysaccharide lyases are the products of various microorganisms, bacteriophage and some eukaryotes. All such enzymes cleave a hexose-1,4-α- or β-uronic acid sequence by β-elimination. They are in some examples, the only known type of enzymes degrading their polyanionic substrates. Although only a small number of these enzymes have been exhaustively studied, the pectin lyases of bacterial origin have proved to be of interesting crystal structure containing a parallel β-helix domain. Alginate and heparin lyases may yield products with biotechnological potential.     

Microbial pectic transeliminases

Pectic transeliminases, also known as pectic lyases or pectinases, are involved in the degradation of pectic substances. They have a wide range of applications in food and textile processing. Although Aspergillus and Penicillium spp. produce pectin lyases, bacteria are the major producers of polygalacturonate lyase. The yields of pectic transeliminases are less than other pectinases. Since new applications for pectic transeliminases are emerging, an improved process for the production of these enzymes is necessary.     

The C-S lyases of higher plants. Purification and characterization of homogeneous alliin lyase of leek (Allium porrum)

The characteristic odors of freshly macerated tissue of Allium species such as garlic and onion are due to the action of the enzyme alliin lyase (EC 4.4.1.4) on endogenous S-alkyl-I-cysteine sulfoxides which are present as secondary amino acids yielding volatile sulfur-containing products. Purification and characterization of the alliin lyase of leek ( Allium porrum L.) has been carried out for comparison with the analogous enzymes previously characterized from garlic and onion. The purification involved homogenization, followed by ammonium sulfate fractionation, elution from an hydroxylapatite column, concentration of the active fractions and passage through a concanavalin A-Sepharose 4B affinity column. The purified enzyme was found to be a glycoprotein with a pH optimum for activity of 8.0. Sodium dodecylsulfate-urea polyacrylamide gel electrophoresis gels of the homogeneous leek enzyme showed it consisted of 1 subunit with a molecular weight of 48000. By gel filtration, 2 stable forms of the native enzyme with molecular weights of 386000 and 580000 were found.     

Cloning and Sequence Analysis of Vibrio halioticoli Genes Encoding Three Types of Polyguluronate Lyase

The alginate lyase-coding genes of Vibrio halioticoli IAM 14596^T, which was isolated from the gut of the abalone Haliotis discus hannai, were cloned using plasmid vector pUC 18, and expressed in Escherichia coli. Three alginate lyase-positive clones, pVHB, pVHC, and pVHE, were obtained, and all clones expressed the enzyme activity specific for polyguluronate. Three genes, alyVG1, alyVG2, and alyVG3, encoding polyguluronate lyase were sequenced: alyVG1 from pVHB was composed of a 1056-bp open reading frame (ORF) encoding 352 amino acid residues; alyVG2 gene from pVHC was composed of a 993-bp ORF encoding 331 amino acid residues; and alyVG3 gene from pVHE was composed of a 705-bp ORF encoding 235 amino acid residues. Comparison of nucleotide and deduced amino acid sequences among AlyVG1, AlyVG2, and AlyVG3 revealed low homologies. The identity value between AlyVG1 and AlyVG2 was 18.7%, and that between AlyVG2 and AlyVG3 was 17.0%. A higher identity value (26.0%) was observed between AlyVG1 and AlyVG3. Sequence comparison among known polyguluronate lyases including AlyVG1, AlyVG2, and AlyVG3 also did not reveal an identical region in these sequences. However, AlyVG1 showed the highest identity value (36.2%) and the highest similarity (73.3%) to AlyA from Klebsiella pneumoniae. A consensus region comprising nine amino acid (YFKAGXYXQ) in the carboxy-terminal region previously reported by Mallisard and colleagues was observed only in AlyVG1 and AlyVG2. Received May 7, 1999; accepted September 4, 1999.     

The Interaction of Monofluorofumarate with Adenylosuccinate Lyase

Abstract

Monofluorofumarate was tested as an alternate substrate and inhibitor for adenylosuccinate lyase. Mono-fluorofumarate was found to be a slow reacting substrate when either AMP or AICAR (5-aminoimidazole 4-carboxamide ribonucleotide) were used as substrate acceptor molecules at pH 7.5. There was no indication that monofluorofumarate could induce the inactivation of adenylosuccinate lyase. The initial reaction product when monofluorofumarate was incubated with AMP in the presence of adenylosuccinate lyase has been determined to be 2-fluoro-adenylosuccinate. This molecule lost HF spontaneously, and the subsequent intermediate was rapidly hydrolyzed to oxalacetate and AMP. A similar reaction scheme was also observed when AICAR was utilized as a cosubstrate with monofluorofumarate. The initial reaction rate when 1.0 mM monofluorofumarate and 1.0 mM AMP were used as substrates with adenylosuccinate lyase was only 1.4% of the rate when 1.0 mM fumarate was used. AICAR (1.0 mM) was found to react with monofluorofumarate at 8.9% of the rate that it reacts with fumarate.     

Expression, purification, and characterization of stable, recombinant human adenylosuccinate lyase

The full length human adenylosuccinate lyase gene was generated by a PCR method using a plasmid encoding a truncated human enzyme as template, and was cloned into a pET-14b vector. Human adenylosuccinate lyase was overexpressed in Escherichia coli Rosetta 2(DE3)pLysS as an N-terminal histidine-tagged protein and was purified to homogeneity by a nickel-nitriloacetic acid column at room temperature. The histidine tag was removed from the human enzyme by thrombin digestion and the adenylosuccinate lyase was purified by Sephadex G-100 gel filtration. The histidine-tagged and non-tagged adenylosuccinate lyases exhibit similar values of Vmax and Km for S-AMP. Analytical ultracentrifugation and circular dichroism revealed, respectively, that the histidine-tagged enzyme is in tetrameric form with a molecular weight of 220 kDa and contains predominantly alpha-helical structure. This is the first purification procedure to yield a stable form of human adenylosuccinate lyase. The enzyme is stable for at least 5 days at 25 degrees C, and upon rapid freezing and thawing. Temperature as well as reducing agent (DTT) play critical roles in determining the stability of the human adenylosuccinate lyase.     

The characterization of mutant Bacillus subtilis adenylosuccinate lyases corresponding to severe human adenylosuccinate lyase deficiencies

Adenylosuccinate lyase is a homotetramer that catalyzes two discrete reactions in the de novo synthesis of purines: the cleavage of adenylosuccinate and succinylaminoimidazole carboxamide ribotide (SAICAR). Several point mutations in the gene encoding the enzyme have been implicated in human disease. Bacillus subtilis adenylosuccinate lyase was used as a model system in which mutations were constructed corresponding to those mutations associated with severe human adenylosuccinate lyase deficiency. Site-directed mutagenesis was utilized to construct amino acid substitutions in B. subtilis adenylosuccinate lyase; Met(10), Ile(123), and Thr(367) were replaced by Leu, Trp, and Arg, respectively, and the altered enzymes were expressed in Escherichia coli. These purified enzymes containing amino acid substitutions were found to have substantial catalytic activity and exhibit relatively small changes in their kinetic parameters. The major deviations from the wild-type-like behavior were observed upon biophysical characterization. All of these enzymes with amino acid replacements are associated with marked thermal instability. I123W adenylosuccinate lyase exhibits notable changes in the circular dichroism spectra, and a native gel electrophoresis pattern indicative of some protein aggregation. T367R also exhibits alterations at the quarternary level, as reflected in native gel electrophoresis. Experimental results, combined with homology modeling, suggest that the altered enzymes are primarily structurally impaired. The enzyme instability was found to be lessened by subunit complementation with the wild-type enzyme, under mild conditions; these studies may have implications for the in vivo behavior of adenylosuccinate lyase in heterozygous patients. Residues Met(10), Ile(123), and Thr(367) appear to be located in regions of the enzyme important for maintaining the structural integrity required for a stable, functional enzyme.     

海藻酸裂解酶酶学与基因工程研究进展及应用

海藻中富含海藻酸盐,海藻酸裂解酶降解后产生的寡糖物质具有很强的生物活性及益生作用,酶法降解海藻酸盐的生物降解取代传统的化学降解已日益受到人们的关注,就海藻酸盐降解酶的来源、作用机制、应用效果和影响因素进行了全面综述,阐明了海藻酸盐降解酶的研究具有显著的理论意义和应用价值。     

A structural basis for depolymerization of alginate by polysaccharide lyase family-7

Alginate lyases depolymerize alginate, a heteropolysaccharide consisting of alpha-L-guluronate and beta-D-mannuronate, through a beta-elimination reaction. Their structure/function relationships are expected to provide information valuable to future industrial alginate processing and drug design for Pseudomonas aeruginosa alginate biofilm-dependent infection, but much remains unknown. Here, we present the crystal structure at 1.0 A resolution and the results of mutational analysis of Sphingomonas sp. A1 alginate lyase A1-II', which is grouped into the polysaccharide lyase (PL) family-7. The overall structure of A1-II' uses a beta-sandwich fold, and it has a large active cleft covered by two short flexible loops. Comparison with other family PL-7 structures indicated that loop opening is necessary for substrate binding when the catalytic reaction is initiated. In contrast to the disorder in many side-chains on the protein surface, the three adjacent beta-strands at the center of the active cleft are well ordered. This results from hydrogen bond networks and stacking-like associations identical with those in other family PL-7 structures. Disruption of these interactions by site-directed mutagenesis (R146A, E148A, R150A, Q189A, and K280A) makes the protein insoluble or greatly decreases its activity. The A1-II' structure includes two sulfate ions in the active cleft. Ammonium sulfate was a potent inhibitor with a Ki of 2.5 mM, indicating that our structure represents a model of the inhibitory state. Results of mutational analysis and continuous hydrogen bond networks suggest that Arg146, Gln189, His191, and Tyr284 form an active center. Tyr284OH appears particularly crucial to the catalytic reaction, which is supported by sulfate ion binding and the proximity to the C5 and O4 atoms of subsite +1 in the model obtained by energy minimization calculations using tri-mannuronate. The structural basis shown by this study is similar in many respects to that of the family PL-5 enzymes.