Tracking X‐ray‐derived redox changes in crystals of a methylamine dehydrogenase/amicyanin complex using single‐crystal UV/Vis microspectrophotometry

X‐ray exposure during crystallographic data collection can result in unintended redox changes in proteins containing functionally important redox centers. In order to directly monitor X‐ray‐derived redox changes in trapped oxidative half‐reaction intermediates of Paracoccus denitrificans methylamine dehydrogenase, a commercially available single‐crystal UV/Vis microspectrophotometer was installed on‐line at the BioCARS beamline 14‐BM‐C at the Advanced Photon Source, Argonne, USA. Monitoring the redox state of the intermediates during X‐ray exposure permitted the creation of a general multi‐crystal data collection strategy to generate true structures of each redox intermediate.     

Synthesis and Characterization of Some New Quinoxalin-2(1H)one and 2-Methyl-3H-quinazolin-4-one Derivatives Targeting the Onset and Progression of CRC with SAR,Molecular Docking,and ADMET Analyses

The pathogenesis of colorectal cancer is a multifactorial process. Dysbiosis and the overexpression of COX-2 and LDHA are important effectors in the initiation and development of the disease through chromosomal instability, PGE2 biosynthesis, and induction of the Warburg effect, respectively. Herein, we report the in vitro testing of some new quinoxalinone and quinazolinone Schiff’s bases as: antibacterial, COX-2 and LDHA inhibitors, and anticolorectal agents on HCT-116 and LoVo cells. Moreover, molecular docking and SAR analyses were performed to identify the structural features contributing to the biological activities. Among the synthesized molecules, the most active cytotoxic agent, (6d) was also a COX-2 inhibitor. In silico ADMET studies predicted that (6d) would have high Caco-2 permeability, and %HIA (99.58%), with low BBB permeability, zero hepatotoxicity, and zero risk of sudden cardiac arrest, or mutagenicity. Further, (6d) is not a potential P-gp substrate, instead, it is a possible P-gpI and II inhibitor, therefore, it can prevent or reverse the multidrug resistance of the anticancer drugs. Collectively, (6d) can be considered as a promising lead suitable for further optimization to develop anti-CRC agents or glycoproteins inhibitors.     

A new mechanism of acyl group transfer in the reaction catalyzed by D-glyceraldehyde-3-phosphate dehydrogenase

D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) catalyzes the oxidative phosphorylation of its substrate in a two-step reaction. Asa result of the first, oxidativestep, the covalent intermediate where in 3-phosphoglyceroyl moiety is bound to Cys149 of the active center is subjected to nucleophilic attack by inorganic phosphate, but remains resistant to hydrolytic decomposition. This ensures tight coupling of oxidation with phosphorylation in glycolysis. In this article, we present the experimental evidence for the conversion of GAPDH into a form capable of performing the reaction in the absence of inorganic phosphate. The structural basis for this conversion is the oxidation of a cysteine residue (probably Cys 153) into a sulfenic acid derivative under mild conditions to affect the integrity of the essential Cys 149. As a result, an intram olecular transfer of 3-phosphoglyceroyl group from the active center Cys 149 to Cys 153 becomes possible with subsequent hydrolysis of the sulfenyl carboxylate intermediate.     

Biocatalytic reduction system for the production of chiral methyl (R)/(S)-4-bromo-3-hydroxybutyrate

An effective method for producing methyl 4-bromo-3-hydroxybutyrate enantiomers was developed using an engineered protein. Escherichia coli transformant cells containing a mutant β-keto ester reductase (KER-L54Q) from Penicillium citrinum and a cofactor-regeneration enzyme such as glucose dehydrogenase (GDH) or Leifsonia sp. alcohol dehydrogenase (LSADH) were used to produce methyl (S)-4-bromo-3-hydroxybutyrate from methyl 4-bromo-3-oxobutyrate. On the other hand, the production of methyl (R)-4-bromo-3-hydroxybutyrate was achieved by asymmetric reduction of methyl 4-bromo-3-oxobutyrate with a mutant phenylacetaldehyde reductase (PAR-HAR1) from Rhodococcus sp. ST-10.     

Carbon Nanotubes‐Ionic Liquid and Chloropromazine Modified Electrode for Determination of NADH and Fabrication of Ethanol Biosensor

Potential cycling was used for oxidation of chloropromazine and producing an electroactive redox couples which strongly adsorbed on the electrode surface modified with carbon nanotubes and ionic liquid nanocomposite. The modified electrode shows excellent electrocatalytic activity toward NADH oxidation. The differential pulse voltammetry detection provided high sensitivity, 0.5835 A M^?1, low detection limit, 80 nM at concentration range up to 20 μM. An ethanol biosensor was also developed by immobilizing alcohol dehydrogenase enzyme onto nanocomposite. Differential pulse voltammetric detection of ethanol gives linear responses over the concentration range 40 μM–1.5 mM with detection limit 5 μM and sensitivity 1.97 μA mM^?1.     

基于药效团模型的DHODH抑制剂构效关系研究

利用药效团模型研究二氢乳清酸脱氢酶(Dihydroorotate dehydrogenase,DHODH)抑制剂的构效关系,为DHODH抑制剂的虚拟筛选提供新的方法.以31个具有DHODH抑制活性的化合物为训练集化合物,半数抑制浓度(IC50)范围为7~63000 nmol/L,利用Catalyst/HypoGen算法构建DHODH抑制剂药效团模型,通过对训练集化合物多个构象进行叠合,提取药效团特征及三维空间限制构建药效团模型.利用基于CatScramble的交叉验证方法及评价模型对已知活性化合物的活性预测能力,确定较优药效团模型.模型包含1个氢键受体、3个疏水中心,表征了受体配体相互作用时可能发生的氢键相互作用、疏水相互作用和π-π相互作用,4个药效特征在三维空间的排列概括了DHODH抑制剂产生活性的结构特点.所得较优模型对训练集化合物及测试集化合物的计算活性值与实验活性值的相关系数分别为0.8405和0.8788.利用药效团模型对来源于微生物的系列化合物进行虚拟筛选,筛选出59个预测活性较好的化合物,可作为进一步药物研发的候选化合物.     

Norcholanic acids as substrates for recombinant 3β-hydroxysteroid dehydrogenase and progesterone 5β-reductase, enzymes of the 5β-cardenolide biosynthesis

The conversion of 23-nor-5,20(22)E-choladienic acid-3β-ol and other intermediates of the putative norcholanic acid pathway of cardenolide biosynthesis by recombinant 3β-hydroxysteroid dehydrogenase from Digitalis lanata in dehydrogenation and reduction reactions was investigated. 23-nor-4,20(22)E-choladienic acid-3-one was found to be a substrate of recombinant progesterone 5β-reductases from D. lanata and Arabidopsis thaliana. The role of various substrates in cardenolide biosynthesis is discussed.     

Zn[(L)Proline]2: an eligible candidate for the synthesis of xanthenediones in water

An efficient route for the condensation of aldehydes with dimedone in the presence of Zn[(L)Proline]₂ in water has been reported. Hydrophilicity of the catalyst is the main attribute of this reaction. The water‐mediated reaction using catalytic amounts of recyclable catalyst avoids the use of expensive, corrosive reagents and toxic solvents and provides operational simplicity. Moreover, column chromatography and recrystallization of product are not required as the crude products itself are very pure and hence can be used for target‐oriented synthesis on a large scale. Copyright © 2012 John Wiley & Sons, Ltd.     

Assay of Serum Glutamic-Oxaloacetic Transaminase Using Malate Dehydrogenase Preparation Obtained from Streptomyces aureofaciens

Abstract

A modified spectrophotometric method for serum glutamic-oxaloacetic transaminase (SGOT) assay was developed. A crude cell-free extract from Streptomyces aureofaciens which showed a high level of malate dehydrogenase (MDH) activity (E.C. 1.1.1.37) was used as the enzymatic indicator. The lyophilized microbial preparation was used without previous purification and was quite stable under refrigeration for one year. Serum sample assays using both the method utilizing the crude cell extract and an enzymatic commercial kit showed good correlation.     

Synthesis and biomimetic mineralization of l‐proline substituted polyphosphazenes as bulk and nanofiber

This work presents the synthesis of polyphosphazenes bearing L ‐proline methyl ester (ProOMe) and 4‐hydroxy‐l ‐proline methyl ester (HypOMe), aiming for new bioactive polymers for bone repair. The polymers were characterized by ¹H and ³¹P NMR, FTIR, DSC, and TGA. Electrospun fibers were prepared using poly[bis(l ‐proline methyl ester)phosphazene] (PProP), and their potential for biomimetic mineralization, as well as the bulk material, were tested in simulated body fluid (1×SBF). Samples were analyzed between 24 h and 3 weeks of incubation using SEM/EDS and FTIR. After 24 h, spherical and flower‐like shapes of calcium phosphates (CaP) were crystallized on the bulk samples. The nanofibers presented spherical CaP crystals attached to them after 48 h of incubation. The Ca/P molar ratio of the crystals varied from 1.5 to 1.6. According to this study, PProP presents bioactivity in vitro, and its fibers offer sites for CaP nucleation like the collagen fibers in bone. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2013 , 51, 1318–1327