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.

Endocrine correlates of mate choice and promiscuity in females of a socially monogamous avian mating system with alternative male reproductive phenotypes

While our understanding of male reproductive strategies is informed by extensive investigations into endocrine mechanisms, the proximate mechanisms by which females compete for mates and adjust reproduction to social environment remains enigmatic. We set out to uncover endocrine correlates of mate choice, social environment, and reproductive investment in female red-backed fairy-wrens Malurus melanocephalus. In this socially monogamous, yet highly sexually promiscuous species, females experience discrete variation in the phenotype of their mates, which vary in both plumage signals and level of paternal care, and in the composition of their breeding groups, which consist of either the pair alone or with an additional cooperative auxiliary; fe- male investment varies according to these social parameters. We found that androgen, estrogen, and glucorticoid levels varied with reproductive stage, with highest androgen and estrogen concentrations during nest construction and highest corticosterone concentrations during the pre-breeding stage. These stage-dependent patterns did not vary with male phenotype or auxiliary presence, though androgen levels during pre-breeding mate selection were lower in females obtaining red/black mates than those obtaining brown mates. We found no evidence that androgen, estrogen, or corticosterone levels during the fertile period were re- lated to extra-pair young （EPY） frequency. This study demonstrates clear changes in steroid levels with reproductive stage, though it found little support for variation with social environment. We suggest hormonal responsiveness to social factors may be physiologically constrained in ways that are bypassed through exogenous hormone manipulations.

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.

Bovine serum albumin:An efficient and green biocatalyst for the one-pot four-component synthesis of pyrano[2,3-c]pyrazoles

The use of biocatalysts is attracting an increasing amount of attention in chemical catalysis.Here,we have shown that bovine serum albumin（BSA）,a ubiquitous,inexpensive,non-enzymatic transport protein,can serve as an efficient,retrievable catalyst in the one-pot four-component reaction of aryl aldehydes,malononitrile,hydrazine hydrate,and ethyl acetoacetate for the synthesis of pyrano[2,3-c]pyrazoles under mild reaction conditions.The BSA biocatalyst also displayed a high catalytic affinity for acyclic/cyclic ketones to yield the corresponding pyrano[2,3-c]pyrazoles or their spirocyclic variants.The BSA could be used for at least five cycles without serious loss of catalytic activity.This novel,efficient protocol has the merits of high yield,operational simplicity,and a relatively benign environmental impact.Moreover,the method extends the promiscuity of BSA as a biocatalyst.

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。

Contribution of Promiscuous Soybean （Glycine max L.） to Upland Rice-based Cropping Systems in West Africa

A two-year on-farm study was carried out at Eglime in the moist savanna （MS） and Ouake in the dry savanna （DS） of Benin to evaluate the contribution of inoculation of dual-purpose soybean varieties to grain yield of upland NERICA rice fertilized with low N level. In 2005, four dual-purpose, promiscuous soybean varieties （cv. TGx1440-IE; TG×1448-2E; TG×1019-2EB; and TG×1844-18E）, and a popular improved variety （cv. Jupiter） were sown in 12 farmer fields with and without Bradyrhizobium japonicum inoculation. There was also land which was left fallow that acted as the control. In 2006, upland interspecific rice （NERICA 1） was sown in all the plots and supplied with 15 kg N haL. Dry matter yield, N accumulation, and net N-balance were significantly enhanced by over 40% with inoculation of cv. TG× 1844-18E than non-inoculation in the DS in comparison to other cultivars. There were no significant effects of inoculation of previous soybean cultivars on soybean grain yield and on the succeeding NERICA rice yield. Averaged over inoculation, previous cv. TG× 1019-2EB plots supplied with only 15 kg N hal gave the highest grain yield, more than twice the yield of control plots in the DS, possibly because of significant production of higher tillers, panicles and harvest index than the other cultivars; and it could be recommended for upland rice-based system for NERICA production.

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。

Financial Value of Nitrogen Fixation in Soybean in Africa： Increasing Benefits for Smallholder Farmers

The knowledge that soil microorganisms form an important component of below ground biodiversity, providing ecosystem services, is often not incorporated in formulation of policies to conserve and manage these microorganisms. Using the method of cost replacement or cost savings in terms of mineral nitrogen fertilizer that would have been required to attain the same level of nitrogen fixed biologically, this study contributes to awareness on the importance of these microorganisms. Applying the knowledge gained from several on-station and on-farm trials in Africa, complemented with assumptions on FAO-sourced data from 19 African countries, this study estimated the financial value of nitrogen fixation of legume nodulating bacteria （LNB） associated with promiscuous soybean varieties. Results show that the financial value of the nitrogen-fixing attribute of soybean in Africa, especially the promiscuous varieties, annually amounts to about $ 200 million US dollars across the 19 countries. With the fertilizer price of -$ 795 tl （June 2008）, this would amount to $ 375 million. The study recommends various ways of increasing the chances of smallholder farmers benefiting from the nitrogen-fixing attribute of LNB, especially since many cannot afford adequate quantities of inorganic fertilizers for increased crop productivity.

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.

Improvement of saccharification of native grass,Pennisetum sp.using cellulase from isolated Aspergillus fumigatus for bioethanol production:an insight into in silico molecular modelling,docking and dynamics studies

Lignocellulosic grass biomass is potential substrate for economical and sustainable bioethanol production.However,the processing cost of bioethanol that majorly includes the hydrolysis of cellulose by cellulases is still a major concern for its industrial production.Thus,knowledge on the sequence to the structural study of cellulase enzyme with consideration of its catalytic region can give important information for effective enzyme engineering and consequently towards enhanced bioethanol production from Pennisetum sp.Therefore,in this study,sequence conservativeness of different cellulosic site among a group of endoglucanase family of cellulase from previously isolated Aspergillus species has been determined.Furthermore,comparative molecular modeling of the endoglucanase from eight different Aspergillus species including Aspergillus fumigatus was conducted and the obtained structures revealed a high degree of difference in their conformational folds.Analysis from InterProScan revealed that the modeled endoglucanase has similar types of domains and share homology with protein family,such as glycoside hydrolase family-61 and fungal cellulose binding domain.Furthermore,molecular docking and interaction studies demonstrated the presence of residues in the endoglucanase of A.fumigatus viz.His20,His88,Asp96,Ala99,Ser100,Ser101,His102,His169,Glu170,Arg173,Glu178,and Tyr218 that are responsible in forming the substrate interaction.An interesting molecular phenomenon,i.e.,catalytic promiscuity has been noted for all the substrate bound complexes of A.fumigatus endoglucanase which also depicts the degree of ligand binding efficacy of the studied enzyme.The molecular interaction study,binding energy analysis and molecular dynamics simulation,demonstrated that heteromeric substrate XylGlc3 is more strongly interacting with the receptor enzyme.Overall,the present findings revealed that important amino acid residues can help in increasing the specificity of endoglucanase from A.fumigatus towards hydrolysis of Pennisetum sp.and other biomass that has an adequate amount of XylGlc3,for possible industrial applications.

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.

Biocatalytic direct asymmetric aldol reaction using proteinase from Aspergillus melleus

The direct asymmetric aldol reaction of aromatic aldehydes with cyclic or acyclic ketones was catalyzed by proteinase from Aspergillus melleus (AMP) in acetonitrile in the presence of water. A wide range of substrates could be transformed into the corresponding aldol products in yields up to 89%, enantioselectivities up to 91% ee and diastereoselectivities up to >99:1 (anti/syn). This work provided an example of enzyme catalytic promiscuity that widens the applicability of this biocatalyst in organic synthesis without the need for additional cofactors or special equipment.