Molecular characterization and structure basis of a malonyltransferase with both substrate promiscuity and catalytic regiospecificity from Cistanche tubulosa

Enzymatic malonylation of natural glycosides provides a promising alternative method for drug-like malonylated glycosides supply.However,the catalytic potential and structural basis of plant malonyltransferase are far from being fully elucidated.This work identified a new malonyltransferase CtMaT1 from Cistanche tubulosa.It displayed unprecedented mono-and/or di-malonylation activity toward diverse glucosides with different aglycons.A“one-pot”system by CtMaT1 and a malonyl-CoA synthetase was established to biosynthesize nine new malonylated glucosides.Structural investigations revealed that CtMaT1 possesses an adequately spacious acyl-acceptor pocket capable of accommodating diverse glucosides.Additionally,it recognizes malonyl-CoA through strong electrotactic and hydrogen interactions.QM/MM calculation revealed the H167-mediated SN2 reaction mechanism of CtMaT1,while dynamic simulations detected the formation of stable hydrogen bonds between the glucose-6-OH group and H167,resulting in its high malonylation regiospecificity.Calculated energy profiles of two isomeric glycosides highlighted lower reaction energy barriers towards glucoside substrates,emphasizing CtMaT1's preference for glucosides.Furthermore,a mutant CtMaT1H36A with notably increased di-malonylation activity was obtained.The underlying molecular mechanism was illuminated through MM/GBSA binding free energy calculation.This study significantly advances the understanding of plant acyltransferases from both functional and protein structural perspectives,while also providing a versatile tool for enzymatic malonylation applications in pharmacology.

Enzymatic Promiscuity： Escherichia coli BioH Esterase-catalysed Aldol Reaction and Knoevenagel Reaction

Esterase BioH,which is obligatory for biotin synthesis in Escherichia coli,was found to exhibit a promiscuous ability to catalyse Aldol and Knoevenagel reactions with moderate to good yields.The reaction conditions including organic solvent,molar ratio of ketone to aldehyde,enzyme amount,and reaction time were investigated to evaluate the effect of different reaction conditions on yield.Target compounds were afforded in the best yield of 91.2％ for Aldol reaction and 54.7％ for Knoevenagel reaction.In addition,because the enzyme could be prepared with a low cost,this protocol could provide an economic route to conduct Aldol and Knoevenagel reactions,which expand the field of enzymatic promiscuity.

Application of Enzymatic Promiscuity in Pharmaceutical Synthesis:Papain-catalyzed One-pot Synthesis of 1,4-Dihydropyridine Calcium Channel Antagonists and Derivatives

A new method for the synthesis of 1,4-dihydropyridine(1,4-DHP)calcium channel antagonists felodipme, nitrendipine and their derivatives via papain-catalyzed three-component reactions of aldehyde,methyl acetoacetate and ethyl 3-aminocrotonate was developed.Operational simplicity,mild reaction conditions and eco-friendliness are the key features of this protocol.

Enzymatic Promiscuity： ＂Amano＂ Lipase AS-catalysed Synthesis of Naphthopyran Derivatives in Anhydrous Media

＂Amano＂ lipase AS（lipase from Aspergillus niger）, which naturally hydrolyzes triglycerides, was found promiscuously to catalyze multi-component reactions of aromatic aldehydes with malononitrile and β-naphthol to prepare naphthopyran derivatives in anhydrous organic solvents in moderate to good yields.

Sniffer在Windows2000下的实现方法

介绍了网络嗅探器 (Sniffer)的基本原理 ,并阐述了Windows 2 0 0 0下Sniffer的基本实现 ,重点阐述了Windows2 0 0

ARP伪装广播发现嗅探器的实现过程分析

目前企业的安全威胁主要来自内部,而且其破坏性也远大于外。其中网络嗅探对于一般的企业网络来说,操作简单同时威胁巨大,很多黑客也使用嗅探器进行网络入侵的渗透,网络嗅探器对信息安全的威胁来自其被动性和非干扰性,使得网络嗅探具有很强的隐蔽性,往往让网络信息泄密变得不容易被发现。

ARP伪装广播发现嗅探器的实现过程分析

目前企业的安全威胁主要来自内部,而且其破坏性也远大于外。其中网络嗅探对于一般的企业网络来说,操作简单同时威胁巨大,很多黑客也使用嗅探器进行网络入侵的渗透,网络嗅探器对信息安全的威胁来自其被动性和非干扰性,使得网络嗅探具有很强的隐蔽性,往往让网络信息泄密变得不容易被发现。

Mutations in FMN binding pocket diminish chromate reduction rates for Gh-ChrR isolated from <i>Gluconacetobacter hansenii</i>

A putative chromate ion binding site was identified proximal to a rigidly bound FMN from electron densities in the crystal structure of the quinone reductase from Gluconacetobacter hansenii (Gh-ChrR) (3s2y.pdb). To clarify the location of the chromate binding site, and to understand the role of FMN in the NADPH-dependent reduction of chromate, we have expressed and purified four mutant enzymes involving the site-specific substitution of individual side chains within the FMN binding pocket that form non-covalent bonds with the ribityl phosphate (i.e., S15A and R17A in loop 1 between β1 sheet and α1 helix) or the isoalloxanzine ring (E83A or Y84A in loop 4 between the β3 sheet and α4 helix). Mutations that selectively disrupt hydrogen bonds between either the N3 nitrogen on the isoalloxanzine ring (i.e., E83) or the ribitylphos- phoate (i.e., S15) respectively result in 50% or 70% reductions in catalytic rates of chromate reduction. In comparison, mutations that disrupt π-π ring stacking interactions with the isoal-loxanzine ring (i.e., Y84) or a salt bridge with the ribityl phosphate result in 87% and 97% inhibittion. In all cases there are minimal alterations in chromate binding affinities. Collectively, these results support the hypothesis that chromate binds proximal to FMN, and implicate a structural role for FMN positioning for optimal chromate reduction rates. As side chains proximal to the β3/α4 FMN binding loop 4 contribute to both NADH and metal ion binding, we propose a model in which structural changes around the FMN binding pocket couples to both chromate and NADH binding sites.

使用PVLAN设计有利于信息化教学的校园网

很多校园网因规划设计不合理而导致其对信息化教学的服务程度不高,校园网应不仅仅只提供简单的上网功能,更需要把各种信息化功能的服务器高速无缝地融合到校园网中,为此提出在交换机上使用PVLAN(Private VLAN)技术。根据校园功能区不同划分校园网,使用PVLAN中的Community、Isolated VLAN特点来进一步细化VLAN划分和数据的流控,借助Promiscuous端口模式的特性,灵活接入各种服务器,给师生的教学、学习、办公、实训提供快捷、安全和全方位的信息化服务。

A fungal CYP from Beauveria bassiana with promiscuous steroid hydroxylation capabilities

Hydroxylation of steroid core is critical to the synthesis of steroid drugs.Direct sp^(3) C-H hydroxylation is challenging through chemical catalysis,alternatively,fungal biotransformation offers a possible solution to this problem.However,mining and metabolic engineering of cytochrome P450 monooxygenases(CYPs)is usually regarded as a more eco-friendly and efficient strategy.Herein,we report the mining and identification of a new steroid CYP(CYP68BE1)from Beauveria bassiana by transcriptomics,heterologous expression,in vivo and in vitro functional characterization.The catalytic promiscuity of CYP68BE1 was explored,and CYP68BE1 showed promiscuously and catalytically versatile,which is qualified for monohydroxylation on C11α,C1α,C6βand dihydroxylation on C1β,11αand C6β,11αof six steroids,leading to the production of key steroid intermediates required in the industrial synthesis of some indispensable steroid drugs.Molecular dynamics simulations were performed,revealing the molecular basis of different binding orientations of CYP68BE1 with different substrates.The discovery of CYP68BE1 offers a promising biocatalyst for enriching the steroid structural and functional diversity,which also can be applied to biosynthesize valuable steroid drug intermediates.

Structural diversification of bioactive bibenzyls through modular co-culture leading to the discovery of a novel neuroprotective agent

Bibenzyls,a kind of important plant polyphenols,have attracted growing attention for their broad and remarkable pharmacological activities.However,due to the low abundance in nature,uncontrollable and environmentally unfriendly chemical synthesis processes,these compounds are not readily accessible.Herein,one high-yield bibenzyl backbone-producing Escherichia coli strain was constructed by using a highly active and substrate-promiscuous bibenzyl synthase identified from Dendrobium officinale in combination with starter and extender biosynthetic enzymes.Three types of efficiently postmodifying modular strains were engineered by employing methyltransferases,prenyltransferase,and glycosyltransferase with high activity and substrate tolerance together with their corresponding donor biosynthetic modules.Structurally different bibenzyl derivatives were tandemly and/or divergently synthesized by co-culture engineering in various combination modes.Especially,a prenylated bibenzyl derivative(12)was found to be an antioxidant that exhibited potent neuroprotective activity in the cellular and rat models of ischemia stroke.RNA-seq,quantitative RT-PCR,and Western-blot analysis demonstrated that 12 could up-regulate the expression level of an apoptosis-inducing factor,mitochondria associated 3(Aifm3),suggesting that Aifm3 might be a new target in ischemic stroke therapy.This study provides a flexible plug-and-play strategy for the easy-to-implement synthesis of structurally diverse bibenzyls through a modular co-culture engineering pipeline for drug discovery.

Plant cytochrome P450 plasticity and evolution

The superfamily of cytochrome P450(CYP)enzymes plays key roles in plant evolution and metabolic diversification.This review provides a status on the CYP Iandscape within green algae and land plants.The 11 conserved CYP clans known from vascular plants are all present in green algae and several green algaespecific clans are recognized.Clan 71,72,and 85 remain the largest CYP clans and include many taxaspecific CYP(sub)families reflecting emergence of linage-specific pathways.Molecular features and dynamics of CYP plasticity and evolution are discussed and exemplified by selected biosynthetic pathways.High substrate promiscuity is commonly observed for CYPs from large families,favoring retention of gene duplicates and neofunctionalization,thus seeding acquisition of new functions.Elucidation of biosynthetic pathways producing metabolites with sporadic distribution across plant phylogeny reveals multiple exampies of convergent evolution where CYPs have been independently recruited from the same or different CYP families,to adapt to similar environmental challenges or ecological niches.Sometimes only a single or a few mutations are required for functional interconversion.A compilation of functionally characterized plant CYPs is provided online through the Plant P450 Database(erda.dk/public/vgrid/PlantP450/).

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

Enzymatic glycosylation catalyzed by glycosyltransferases (GTs) has great potential in creating diverse novel and bioactive glycosides. Herein, three new GTs (UGT84 A33, UGT71 AE1 and UGT90 A14) from Carthamus tinctorius exhibited robust catalytic promiscuity to benzylisoquinoline alkaloids, and were used as enzymatic tools in glycosylation of bioactive benzylisoquinoline alkaloids. Seven novel benzylisoquinoline alkaloids O-glycosides were synthesized with high efficiency. These studies indicate the significant potential of promiscuous GTs in synthesis of benzylisoquinoline alkaloids glycosides for drug discovery.

UGT88B2: A promiscuous O-glycosyltransferase from Carthamus tinctorius

Objective:In order to obtain new glycosyltransferases with highly efficient catalysis,the glycosyltransferases from Carthamus tinctorius which contains diverse types of glycosides were mined.Methods:A new glycosyltransferase gene(UGT88B2)with full length was obtained by PCR and further transformed into Escherichia coli for heterologous expression.The catalytic activity of recombinant UGT88B2 was determined by HPLC-MSn.The structures of representative catalytic products were elucidated by MS and NMR.Results:UGT88B2 exhibited catalytic promiscuity and various patterns in glycosylation of flavonoids with high efficiency.Conclusion:A new glycosyltransferase named UGT88B2 was successfully mined and can be employed as enzymatic tools in glycosylation of flavonoids.

Linux的网络流量监测

本文旨在介绍基于Linux的网络流量监测系统,本系统是一个基于TCP/IP协议的网络应用程序,它提供了一个能够对局域网内部网络数据流量的详尽统计的有用工具,为网络管理提供了重要手段。其使用对象为各学校、科研单位、政府部门、公司企业的网络管理中心。该系统通过对以太网数据包的获取、分析、处理,即对Arp包、IP包的收集、分析,对IP包协议TCP、UDP、ICMP的分析、处理,从而达到对网络流量的监测和统计。

Enzyme-catalyzed asymmetric domino aza-Michael/aldol reaction for the synthesis of 1,2-dihydroquinolines using pepsin from porcine gastric mucosa

An unprecedented enzyme-catalyzed asymmetric domino aza-Michael/aldol reaction of 2-aminobenzaldehyde and a,b-unsaturated aldehydes is achieved. Pepsin from porcine gastric mucosa provided mild and efficient access to diverse substituted 1,2-dihydroquinolines in yields of 38%–97% with 6%–24%enantiomeric excess（ee）. This work not only provides a novel method for the synthesis of dihydroquinoline derivatives, but also promotes the development of enzyme catalytic promiscuity.

Advances in photo-enzymatic-coupling catalysis system

Photosynthesis,as an efficient pathway for solar energy capture and utilization,has supported aerobionts for billions of years.The imitation of photosynthesis to construct artificial photo-enzymatic-coupling catalysis system has become a pow-erful means to solve energy and environmental problems.After years of in-depth research on this coupled system,through ingenious and rational design,the synergistic effect of photo-and enzymatic catalyses has played a significant role in many different fields,including solar-driven fuel production,chiral chemical synthesis and carbon dioxide fixation.Furthermore,light in enzymatic catalysis could also endow enzyme new possibilities.Photo-induced radical cofactor could bring catalytic promiscuity to enzymes,making them catalyze reactions that natural enzymes cannot.This review summarizes the advances in photo-enzymatic-coupling catalysis system and introduces its essential components,their integration and application.The possibilities presented by photo-induced catalytic promiscuity and its significance for expanding the toolbox of enzymes are also discussed.

Genome Mining and Biosynthesis Study of a Type B Linaridin Reveals a Highly Versatileα-N-Methyltransferase

Linaridins are a small but growing family of natural products belonging to the ribosomally synthesized and post-translationally modified peptide(RiPP)superfamily.In this study,a genome mining approach led to the identification of a novel linaridin,mononaridin(MON),from Streptomyces monomycini.In-frame deletion genetic knockout studies showed that,in addition to many genes essential for MON biosynthesis,monM encodes an S-adenosyl methionine(SAM)-dependentα-N-methyltransferase that is responsible for installing two methyl groups in the MON N-terminus.Besides SAM,MonM also accepts ethyl-SAM and allyl-SAM,in which the methyl of SAM is replaced by an ethyl and an allyl,respectively.We showed that ethyl-SAM and allyl-SAM have distinct reactivities in MonM catalysis,and this observation was further investigated in detail by density functional theory(DFT)calculations.Remarkably,MonM acts efficiently on nisin,a prototypic lantibiotic that is structurally very different from the native substrate,and the ability of MonM to transfer an allyl group to the nisin N-terminus allowed production of a fluorescently labeled nisin,which can be further used in microscopic cell analysis.Our studies provide new insights into linaridin biosynthesis and demonstrate the potential of linaridin methyltransferases in bioengineering applications.

Consecutive Methylation Catalyzed by TsrM,an Atypical Class B Radical SAM Methylase

TsrM is a cobalamin-dependent radical S-adenosylmethionine(SAM)methyltransferase belonging to the Class B radical SAM methylase(RSM)family.This enzyme catalyzes the C-2 methylation of L-tryptophan to produce 2-methyltrytophan(2-MeTrp),an intermediate involved in the biosynthesis of thiostrepton A.In this work,we report characterization of an unexpected activity of TsrM,which carries out an additional methylation reaction on the product 2-MeTrp.A series of isotopic labeling studies and assays with different Trp analogs revealed that TsrM is able to transfer a methyl group from SAM to the C4 of 2-MeTrp to produce 2,4-dimethyltryptophan.These results reveal the intriguing substrate specificity of TsrM,further expanding the reaction promiscuity of the radical SAM superfamily enzymes.

‘‘Amano'' lipase DF-catalyzed efficient synthesis of 2,20-arylmethylene dicyclohexane-1,3-dione derivatives in anhydrous media

A simple and efficient method was developed for the synthesis of 2.2＇-arylmethylene dicyclohexane-1,3- dione derivatives via the Knoevenagel-Michael cascade reactions of aromatic aldehydes and 1,3-cyclic diketones catalyzed by ＂Amano＂ lipase DF, which expands the application field of enzyme catalytic promiscuity. This protocol provides several advantages over the traditional chemical synthesis, such as simple work-up procedure, high yields Cup to 94%） and environmental friendliness.