环果寡糖果糖基转移酶研究进展

环果寡糖果糖基转移酶(CFT酶)是一种多功能型酶,能催化三种转糖基反应(环化、歧化和耦合)以及水解反应,而其中能将菊糖水解为环果寡糖的环化反应是其特征性反应。环果寡糖的独特结构使其在医药、化工等领域具有潜在应用价值。简要论述了环果寡糖的结构和应用,重点论述了环果寡糖果糖基转移酶的酶学性质、基因克隆和表达,以及功能和催化机理等方面的最新研究进展。     

催化混杂性驱动的酶功能重塑

作为生物催化剂,酶蛋白介导的生化反应具有条件温和、绿色环保等优点。然而相比化学催化剂,天然酶功能的局限性制约了它在生物制造领域的广泛应用。前期研究表明,酶蛋白除了催化专一性外,同时还展现出混杂性的一面,可在特定条件下催化非天然模式反应。这一特性为酶分子功能重塑提供了新思路,可用来指导人工酶设计,拓展天然酶的催化边界,实现新颖酶促反应类型,以扩大酶催化应用场景。本文从酶催化功能混杂性背后可能的进化机制入手,综述了当前诱导酶催化功能混杂性的常用策略,如定向进化、构象动力学、反应条件诱导及祖先酶重构等技术,并从催化机制、构效关系及适应性进化等多个角度,结合近年来相关研究实例,探讨了催化功能混杂性背后的分子机制,为突破天然酶促反应局限性、创制催化非天然反应的高效人工酶元件提供参考。     

环糊精葡萄糖基转移酶的结构特征与催化机理

随着环糊精在食品、医药等领域的应用越来越广,生产环糊精所必需的环糊精葡萄糖基转移酶(CGT酶)已经成为当今研究的热点。特别是近二十年来,国外对该酶进行了比较深入的研究。首先介绍了CGT酶的功能特性与结构特征。CGT酶是一种多功能型酶,能催化三种转糖基反应(歧化、环化和耦合反应)和水解反应,其中,能将淀粉转化为环糊精的环化反应是特征反应;作为α-淀粉酶家族的成员,CGT酶除了具有与α-淀粉酶相同的A、B、C结构域外,还存在D和E结构域。另外,对CGT酶的催化机理包括底物结合方式、转糖苷反应机理以及环化机理等进行了详细的讨论。     

基于序列和结构分析的酶热稳定性改造策略*

功能酶被广泛应用于食品、化工、医药等领域,但却容易受高温环境限制,导致催化效率降低。以分子改造为目的的蛋白质工程技术是解决这一问题的关键环节,其能够对酶结构和功能进行改造,获得热稳定性好的工业酶。传统的定向进化方法只能依靠随机突变进行人工筛选,具有效率低、针对性差等缺点;理性设计作为酶热稳定性改造的主要方法,可借助各种计算机程序和软件预测潜在突变位点,但其要求对酶的催化机制、热稳定性机制有深入了解。对于大多数天然酶而言,酶的序列和晶体结构是最容易获取的信息,也是预测功能的重要基础。从酶的序列和晶体结构入手,重点介绍了共识突变、基于序列偏好性的突变、截短柔性区域、优化分子内相互作用力、刚化催化活性区域及计算机辅助筛选柔性位点等常用策略,这些策略具有筛选效率高、改造准确性高、实用性强等优点。结合多种酶的热稳定性改造案例进行分析,旨在为不同酶的改造策略选择提供有效参考,同时也为工业酶的耐热性研究提供理论支持。     

抗体酶技术在医学上的应用

抗体酶是一类可变区被赋予了酶催化活性的抗体分子,可以催化许多天然酶无法催化的化学反应,在医学、工业、农业等众多领域具有广泛的应用前景和潜在的应用价值。该文介绍抗体酶及其制备策略和方法;抗体酶技术在医学领域的应用及研究进展。     

环氧角鲨烯环化酶在三萜化合物生物合成中的进展

三萜类化合物是植物代谢产物中最具多样性的化合物之一,具有广泛的生理活性和重要的经济价值.环氧角鲨烯环化酶(oxidosqualene cyclases,OSCs)催化2,3-氧化鲨烯环化生成不同类型的甾醇和植物三萜化合物,对天然产物的结构多样性具有重要意义.然而,目前对于OSCs酶催化2,3-氧化鲨烯发生环化多样性的机...     

结构修饰提高酶稳定性、活性

酶因其特异性和可持续性而成为广泛应用的绿色催化剂，其稳定性和催化活性是决定酶适用性的关键因素。为满足实际应用需求，通过蛋白质结构修饰赋予其所需的催化特性是当前的研究热点。提高热稳定性的策略有：引入非共价/共价相互作用（疏水相互作用、氢键、盐桥、芳香环相互作用、二硫键）、环截短、C端和N端工程，及增加脯氨酸/减少甘氨酸的数目等；获得具有高效性和多样性的生物催化剂的策略有：降低空间位阻、拓宽催化口袋、增加底物亲和力及调节活性位点灵活性等。然而，在稳定性或催化功能改造的过程中，新突变的引入会削弱其他功能，致使进化过程中稳定性和催化活性相互制约。因此，采用基于理性计算优选突变热点、基于多重蛋白质稳定性或活性改造策略的共进化，以及基于高度稳定的蛋白质骨架创造或/和优化蛋白质功能等多种策略克服酶稳定性-活性之间的权衡。本综述重点阐述了结构修饰方法在提高酶稳定性或/和催化活性方面的应用，并展望了该领域的未来发展前景。     

蛋白质交联用酶的作用机制及研究进展北大核心CSCD

蛋白质交联在食品、化工和医药等领域发挥重要的作用。利用酶催化蛋白质交联是一种可替代物理和化学交联的高效经济的方法。然而,目前仍缺乏详细的酶促蛋白质交联分子层面的解析。本文综述了酶催化的蛋白质交联反应机制及其对蛋白质结构的影响,以及在食品、化工和医药领域的应用,并展望了酶促交联的发展。     

微生物降解有机磷类毒剂的酶学研究进展*

有机磷毒剂在农业生产和战争中的应用广泛,其生物解毒受到理论和应用研究方面的重视。有机磷水解酶在有机磷毒剂的生物降解中具有重要的作用,目前在有机磷水解酶的蛋白质晶体结构、酶促反应机制等方面的研究取得了较大的进展。中对有机磷水解酶在酶学、高级结构、催化机制、定向进化及其应用等方面的研究进行了综述,并对该领域今后的研究趋势进行了展望。     

纳米酶:新一代人工模拟酶

正酶是活细胞产生的一类具有催化作用的有机分子.1877年,德国科学家Wilhelm Kuhne将这类分子命名为"酶".随后,美国科学家James B.Sumner将其鉴定为一种蛋白质.天然酶具有催化效率高、底物专一、反应条件温和等特点.然而,由于酶的化学本质是蛋白质,在酸、碱、热等非生理环境中容易发生结构变化而失活.为此,科学家一直在寻求用化学合成法制备人工模拟酶,以便在非生理环境中应用.但如何提高模拟酶的催化效率,一直是该领域的核心问题之一.特别是,能否用无机材料制造出人工酶,使其不仅具有生物酶     

Stabilization of a binary protein complex by intein-mediated cyclization

The study of protein-protein interactions can be hampered by the instability of one or more of the protein complex components. In this study, we showed that intein-mediated cyclization can be used to engineer an artificial intramolecular cyclic protein complex between two interacting proteins: the largely unstable LIM-only protein 4 (LMO4) and an unstructured domain of LIM domain binding protein 1 (ldb1). The X-ray structure of the cyclic complex is identical to noncyclized versions of the complex. Chemical and thermal denaturation assays using intrinsic tryptophan fluorescence and dynamic light scattering were used to compare the relative stabilities of the cyclized complex, the intermolecular (or free) complex, and two linear versions of the intramolecular complex (in which the interacting domains of LMO4 and ldb1 were fused, via a flexible linker, in either orientation). In terms of resistance to denaturation, the cyclic complex is the most stable variant and the intermolecular complex is the least stable; however, the two linear intramolecular variants show significant differences in stability. These differences appear to be related to the relative contact order (the average distance in sequence between residues that make contacts within a structure) of key binding residues at the interface of the two proteins. Thus, the restriction of the more stable component of a complex may enhance stability to a greater extent than restraining less stable components.     

Characterization of wild-type and mutant forms of human tryptophan hydroxylase 2

Tryptophan hydroxylase (TPH) catalyses the rate-limiting step in the biosynthesis of serotonin. In vertebrates, the homologous genes tph1 and tph2 encode two different enzymes with distinct patterns of expression, enzyme kinetics and regulation. Variants of TPH2 have recently reported to be associated with reduced serotonin production and behavioural alterations in man and mice. We have produced the human forms of these enzymes in Esherichia coli and in human embryonic kidney cell lines (HEK293) and examined the effects of mutations on their heterologous expression levels, solubility, thermal stability, secondary structure, and catalytic properties. Pure human TPH2 P449R (corresponds to mouse P447R) had comparable catalytic activity (V(max)) and solubility relative to the wild type, but had decreased thermal stability; whereas human TPH2 R441H had decreased activity, solubility and stability. Thus, we consider the variations in kinetic values between wild-type and TPH2 mutants to be of secondary importance to their effects on protein stability and solubility. These findings provide potential molecular explanations for disorders related to the central serotonergic system, such as depression or suicidal behaviour.     

A comparative infrared spectroscopic study of glycoside hydrolases from extremophilic archaea revealed different molecular mechanisms of adaptation to high temperatures

The identification of the determinants of protein thermal stabilization is often pursued by comparing enzymes from hyperthermophiles with their mesophilic counterparts while direct structural comparisons among proteins and enzymes from hyperthermophiles are rather uncommon. Here, oligomeric beta-glycosidases from the hyperthermophilic archaea Sulfolobus solfataricus (Ss beta-gly), Thermosphaera aggregans (Ta beta-gly), and Pyrococcus furiosus (Pf beta-gly), have been compared. Studies of FTIR spectroscopy and kinetics of thermal inactivation showed that the three enzymes had similar secondary structure composition, but Ss beta-gly and Ta beta-gly (temperatures of melting 98.1 and 98.4 degrees C, respectively) were less stable than Pf beta-gly, which maintained its secondary structure even at 99.5 degrees C. The thermal denaturation of Pf beta-gly, followed in the presence of SDS, suggested that this enzyme is stabilized by hydrophobic interactions. A detailed inspection of the 3D-structures of these enzymes supported the experimental results: Ss beta-gly and Ta beta-gly are stabilized by a combination of ion-pairs networks and intrasubunit S-S bridges while the increased stability of Pf beta-gly resides in a more compact protein core. The different strategies of protein stabilization give experimental support to recent theories on thermophilic adaptation and suggest that different stabilization strategies could have been adopted among archaea.     

The effect of cyclization on the enzymatic degradation of herpes simplex virus glycoprotein D derived epitope peptide.

One linear and three cyclic peptides corresponding to the 278-287 ((278)LLEDPVGTVA(287)) sequence of glycoprotein D (gD-1) of herpes simplex virus were synthesized for the analysis of the effect of cyclization on protection against enzymatic degradation. In this design, the turn-forming motif ((281)DPVG(284)) was positioned in the central part of the peptide and elongated by three amino acids at both termini. Cyclopeptide formation was achieved by the introduction of a peptide bond, a disulfide bridge or a thioether link. The stability of these peptides was compared in human serum and also in rat lysosomal preparations. The data obtained in 10% and 50% human serum show that all three types of cyclization enhanced the stability, but at different levels. Complete stability was only achieved by the introduction of a thioether link, while the presence of a disulfide or peptide bond resulted in improved, but partial resistance against hydrolytic decomposition. In lysosomal preparations the presence of cyclic primary structure provided full protection against enzymatic hydrolysis. Taken together, these findings indicate that by appropriate structural modification it is feasible to construct a synthetic antigen with high stability against enzymatic degradation in complex biological fluids. Further studies are in progress to identify enzymes responsible for degradation in diluted human sera as well as in the lysosomal preparations and to gain more detailed information on the mechanism of action.     

Structure, mechanism and function of prenyltransferases.

In this review, we summarize recent progress in studying three main classes of prenyltransferases: (a) isoprenyl pyrophosphate synthases (IPPSs), which catalyze chain elongation of allylic pyrophosphate substrates via consecutive condensation reactions with isopentenyl pyrophosphate (IPP) to generate linear polymers with defined chain lengths; (b) protein prenyltransferases, which catalyze the transfer of an isoprenyl pyrophosphate (e.g. farnesyl pyrophosphate) to a protein or a peptide; (c) prenyltransferases, which catalyze the cyclization of isoprenyl pyrophosphates. The prenyltransferase products are widely distributed in nature and serve a variety of important biological functions. The catalytic mechanism deduced from the 3D structure and other biochemical studies of these prenyltransferases as well as how the protein functions are related to their reaction mechanism and structure are discussed. In the IPPS reaction, we focus on the mechanism that controls product chain length and the reaction kinetics of IPP condensation in the cis-type and trans-type enzymes. For protein prenyltransferases, the structures of Ras farnesyltransferase and Rab geranylgeranyltransferase are used to elucidate the reaction mechanism of this group of enzymes. For the enzymes involved in cyclic terpene biosynthesis, the structures and mechanisms of squalene cyclase, 5-epi-aristolochene synthase, pentalenene synthase, and trichodiene synthase are summarized.     

Probing Structural Elements of Thermal Stability in Bacterial Oligomeric Alcohol Dehydrogenases. I. Construction and Characterization of Chimeras Consisting of Secondary ADHs from Thermoanaerobacter brockii and Clostridium beijerinckii

Two tetrameric secondary alcohol dehydrogenases (ADHs), one from the mesophile Clostridium beijerinckii (CBADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TBADH), share 75% sequence identity but differ by 26 °C in thermal stability. To explore the role of linear segments of these similar enzymes in maintaining the thermal stability of the thermostable TBADH, a series of 12 CBadh and TBadh chimeric genes and the two parental wild-type genes were expressed in Escherichia coli, and the enzymes were isolated, purified and characterized. The thermal stability of each chimeric enzyme was approximately exponentially proportional to the content of the amino acid sequence of the thermophilic enzyme, indicating that the amino acid residues contributing to the thermal stability of TBADH are distributed along the whole protein molecule. It is suggested that major structural elements of thermal stability may reside among the nine discrepant amino acid residues between the N-terminal 50-amino acid residues of TBADH and CBADH.     

Discovery and characterization of a linear cyclotide from Viola odorata: implications for the processing of circular proteins

Cyclotides are mini-proteins of 28-37 amino acid residues that have the unusual feature of a head-to-tail cyclic backbone surrounding a cystine knot. This molecular architecture gives the cyclotides heightened resistance to thermal, chemical and enzymatic degradation and has prompted investigations into their use as scaffolds in peptide therapeutics. There are now more than 80 reported cyclotide sequences from plants in the families Rubiaceae, Violaceae and Cucurbitaceae, with a wide variety of biological activities observed. However, potentially limiting the development of cyclotide-based therapeutics is a lack of understanding of the mechanism by which these peptides are cyclized in vivo. Until now, no linear versions of cyclotides have been reported, limiting our understanding of the cyclization mechanism. This study reports the discovery of a naturally occurring linear cyclotide, violacin A, from the plant Viola odorata and discusses the implications for in vivo cyclization of peptides. The elucidation of the cDNA clone of violacin A revealed a point mutation that introduces a stop codon, which inhibits the translation of a key Asn residue that is thought to be required for cyclization. The three-dimensional solution structure of violacin A was determined and found to adopt the cystine knot fold of native cyclotides. Enzymatic stability assays on violacin A indicate that despite an increase in the flexibility of the structure relative to cyclic counterparts, the cystine knot preserves the overall stability of the molecule.     

Sortase A‐mediated synthesis of ligand‐grafted cyclized peptides for modulating a model protein‐protein interaction

Specific ligand‐grafted cyclic peptides are promising drug candidates that can modulate protein‐protein interactions (PPIs) with increased proteolytic stability. In this study, we aimed to demonstrate that Sortase A (SrtA)‐mediated peptide transpeptidation can be applied to produce bioactive sequence‐grafted, stable, cyclic peptides. A naturally occurring cyclic peptide, sunflower trypsin inhibitor 1 (SFTI‐1), was selected as the scaffold, and a tetrapeptide motif, Glu‐Ser‐Asp‐Val (ESDV), was grafted into the scaffold as a model ligand. The linear precursor of the grafted peptide with SrtA‐recognition motifs at the N‐ and C‐termini was cyclized in good yield simply by co‐incubation with SrtA. The ESDV‐grafted cyclic SFTI‐1 obtained was confirmed to have high stability against proteolysis by human serum and bound to the target PDZ2 domain of postsynaptic density‐95 protein. An optimized sequence‐grafted cyclic SFTI‐1 could competitively suppress the interaction of PDZ2 with its natural ligand, the C‐terminal peptide of the NR2B subunit of the N‐methyl‐D‐aspartate receptor. These results show that a strategy combining peptide grafting into the SFTI‐1 scaffold with SrtA‐catalyzed cyclization can be a simple and effective method for producing stable peptide drugs.     

Phage P22 tailspike protein: removal of head-binding domain unmasks effects of folding mutations on native-state thermal stability.

A shortened, recombinant protein comprising residues 109-666 of the tailspike endorhamnosidase of Salmonella phage P22 was purified from Escherichia coli and crystallized. Like the full-length tailspike, the protein lacking the amino-terminal head-binding domain is an SDS-resistant, thermostable trimer. Its fluorescence and circular dichroism spectra indicate native structure. Oligosaccharide binding and endoglycosidase activities of both proteins are identical. A number of tailspike folding mutants have been obtained previously in a genetic approach to protein folding. Two temperature-sensitive-folding (tsf) mutations and the four known global second-site suppressor (su) mutations were introduced into the shortened protein and found to reduce or increase folding yields at high temperature. The mutational effects on folding yields and subunit folding kinetics parallel those observed with the full-length protein. They mirror the in vivo phenotypes and are consistent with the substitutions altering the stability of thermolabile folding intermediates. Because full-length and shortened tailspikes aggregate upon thermal denaturation, and their denaturant-induced unfolding displays hysteresis, kinetics of thermal unfolding were measured to assess the stability of the native proteins. Unfolding of the shortened wild-type protein in the presence of 2% SDS at 71 degrees C occurs at a rate of 9.2 x 10(-4) s(-1). It reflects the second kinetic phase of unfolding of the full-length protein. All six mutations were found to affect the thermal stability of the native protein. Both tsf mutations accelerate thermal unfolding about 10-fold. Two of the su mutations retard thermal unfolding up to 5-fold, while the remaining two mutations accelerate unfolding up to 5-fold. The mutational effects can be rationalized on the background of the recently determined crystal structure of the protein.