Substrate specificity of NovM: implications for novobiocin biosynthesis and glycorandomization

[reaction: see text] In an effort to expand the scope of natural product in vitro glycorandomization (IVG), the substrate specificity of NovM was investigated. A test of four aglycon analogues and over 40 nucleotide sugars revealed NovM has a surprisingly stringent substrate specificity and provided only three new "unnatural" natural products. On the basis of the determined substrate specificity, an alternative to the sugar nucleotide biosynthetic dogma and a cautionary note for the general applicability of IVG are introduced.     

Daptomycin structure and mechanism of action revealed

Daptomycin kills otherwise antibiotic-resistant gram-positive pathogens and is the first lipopeptide antibiotic to reach the clinic. Elucidation of its 3D structure and mechanism of action, reported in this issue of Chemistry & Biology, will facilitate the design and engineering of new, potentially life-saving antibiotics.     

Structure and mechanism of action of isopentenylpyrophosphate-dimethylallylpyrophosphate isomerase

We have obtained the three-dimensional X-ray crystallographic structure of a C67A mutant Escherichia coli isopentenylpyrophosphate-dimethylallylpyrophosphate isomerase (EC 5.3.3.2) complexed with the bromohydrin of isopentenylpyrophosphate, at 1.93 A resolution. The overall backbone fold is very similar to that obtained previously for the wild-type enzyme in the presence of a divalent metal cation (Mn2+ or Mg2+). However, in the new structure, there are two metal binding sites, not just one. The first metal binding site is occupied by Mn2+, coordinated to three histidine and two glutamate residues, while the second is occupied by Mg2+, coordinated to two bromohydrin-ligand phosphate oxygens, the carbonyl oxygen of A67, a carboxyl oxygen of E87, and two water molecules. The C3 hydroxyl group of the bromohydrin inhibitor is involved in a short hydrogen bond to the carboxyl group of E116, one of the two Mn-bound glutamates. The structure obtained is consistent with a mechanism of action of the enzyme in which the carboxyl group of E116 protonates the double bond in isopentenylpyrophosphate, forming a carbocation, followed by removal of a C2 proton by the thiolate of C67, in the wild-type enzyme. The inhibition of the enzyme by a wide variety of other potent inhibitors is also readily explained on the basis of the bromohydrin inhibitor structure.     

高效液相色谱法测定复方秋水仙碱胶囊中秋水仙碱的含量

为建立复方秋水仙碱胶囊中秋水仙碱的含量测定方法 ,采用高效液相色谱法 ,色谱柱Kromasil1 0 0～ 5C18( 5 μm ,4 6mm× 1 5 0mm) ,流动相为V( 0 0 5mol/L磷酸二氢钾 ) +V(甲醇 ) =5 0+5 0 ,流速 1 0mL/min ,紫外检测波长 3 5 0nm ,柱温 2 0℃ ,外标法定量。结果表明 ,秋水仙碱在3 0 5～ 9 1 5 μg/mL之间线性关系良好 ,回归方程为y =47 1 43 0x -1 3 82 7,r =0 9994(n =5 ) ,平均回收率为 1 0 0 1 9% ,RSD =0 86% (n=9)。本法简便、准确、可靠 ,可作为控制复方秋水仙碱胶囊质量标准的方法。     

Specificity and molecular mechanism of abortificient action of prostaglandins

The paper reports comparison of electrostatic charge and energy distribution on the basis of the CNDO /2 method for six forms of prostaglandins–PGF_2α, PGF_1β, PGE₁, PGE₂, PGB₁, and PGA₁–having diverse physiological action. The isopotential mapping done in three dimension showed that the lower value of electrostatic potential and proximity of the two low energy regions around O₉ and O₁₁ in PGE₂ and PGF_2α is probably responsible for their higher abortificient activity. We also compare here the variation of the long- and short-range interaction between ring–chain and chain–chain portion of different forms and compared them with the variation in their action.     

Research on synthesis and action mechanism of polycarboxylate superplasticizer

The relationship between the structure and performance of polycarboxylate superplasticizer was analyzed. The respective functions of the structure units of the main and branched chains were discussed. The progress of synthesis and molecular structure design and synthesis of polycarboxylate superplasticizer were reviewed according to the difference in the structure unit of the main chain. Results indicated that their performance is related to the structure unit of the main and branched chains, as well as the position and species of functional groups. The polycarboxylate superplasticizer, which had suitable graft and block polymers of polyethylene glycol or polyoxyethylene, and a suitable sulfonic group, had small slump loss besides high water-reducing performance. On the other hand, the hydroxyl group at the end of the chain causes gelation easily. On the basis of the items mentioned above, as well as the source and cost of raw materials, esterification of polyethylene glycol and acrylic acid were first adopted using para-toluene sulfonic acid as catalyst, then polymerized with sodium sulfonate methacrylate. A certain amount of acrylic was added in order to regulate both the polymerization degree of the main chain and the ratio of carboxyl and sulfonic groups in the branched chain. As a result, the high performance superplasticizer has been synthesized (was obtained). The divergence of the cement plasma is about 200 mm when the addition amount of superplasticizer is 0.16%–0.20% of cement weight, and the ratio of the water and cement is 0.29. Translated from Journal of Wuhan Uniwersity of Technology, 2006, 28(9): 18–20 [译自: 武汉理工大学学报]     

A noncanonical path to mechanism of action

Improved methods for discovering small-molecule mechanisms of action are needed. In this issue of Chemistry & Biology, Zhang et al. make clever use of the zebrafish to study the mechanism of the angiogenesis inhibitor fumagillin and reveal that it targets the noncanonical Wnt pathway.     

The mechanism of action and place of application of capillary forces

The question of the mechanism of action and the place of application of capillary forces has been discussed. It has been shown that the conventional notions of the character of capillary forces are often contradictory. A molecular-kinetic approach has been employed to determine the places of application of capillary forces and the mechanism of their action.     

Ring-deactivated hydroxyalkylpyrrole-based inhibitors of alpha-chymotrypsin: synthesis and mechanism of action

13C NMR and mass spectrometry studies have been used to demonstrate that the inhibition of alpha-chymotrypsin by N-sulfonylhydroxymethylpyrrole inhibitors (10) is non-covalent. Hydroxyalkylpyrroles in which an electron-withdrawing group (acyl substituent) is introduced at the alternative C2 position have been synthesised and also shown to inactivate alpha-chymotrypsin. SAR studies on this class suggests that the incorporation of phenylalanine at C2 is favoured, however, there is little gain in introducing a hydrophobic substituent at C5.     

Synthesis of Lipid Derivatives of Colchicine

The synthesis of glycero-lipids linked to colchicine derivatives is reported. The lipid structures are designed to perform two-dimensional crystallization experiments with tubulin, the structural subunit protein of microtubules.     

秋水仙碱分析方法的研究概述

针对秋水仙碱作为治疗急性痛风性关节炎药物的应用,综述了国内外秋水仙碱测量方法研究进展,重点介绍了伏安法、高效液相色谱法、高效液相色谱-质谱联用法等的研究情况.     

Simple Conversion of Colchicine into Demecolcine

N-Deacetylcolchiceine ( 7 ), readily available from colchicine ( 1 ), was converted into N-trifluoroacetyl-deacetylcolchiceine ( 8 ). Methylation of 8 with methyl iodide in the presence of potassium carbonate afforded a mixture of N-trifluoroacetyl-demecolcine ( 10 ) and its isomer 11 . The mixture of 10 and 11 was detrifluoroacetylated and separated by chromatography to afford demecolcine ( 2 ) and isodemecolcine ( 12 ). A more practical route to 2 started with 8 , and gave N-trifluoroacetyl-deacetylcolchicine ( 13 ) and its isomer 14 after O-methylation with diazomethane. N-Methylation of 13 and 14 with methyl iodide and potassium carbonate afforded 10 and 11 . The overall yield in the conversion of colchicine ( 1 ) into demecolcine ( 2 ) via 7, 8 and 13 was 55%.     

Thiamine models and perspectives on the mechanism of action of thiamine-dependent enzymes

Thiamine dependent enzymes catalyze ligase and lyase reactions near a carbonyl moiety. Chemical models for these reactions serve as useful tools to substantiate a detailed mechanism of action. This tutorial review covers all such studies performed thus far, emphasizing the role of each part around the active site and the conformation of the cofactor during catalysis.     

Structure-based engineering of E. coli galactokinase as a first step toward in vivo glycorandomization

In vitro glycorandomization is a rapid chemoenzymatic strategy to diversify complex natural product scaffolds. The glycorandomization sugar activation pathway is dependent upon the efficient construction of diverse sugar-1-phosphate libraries. In the context of the previously evolved GalK Y371H "gatekeeper" mutation, the active site M173L mutation described herein presents a kinase with remarkably broadened substrate range to include 28 diverse natural and unnatural sugars. Among these new substrates, 6-azido-6-deoxy-galactose and 6-azido-6-deoxy-glucose present unique chemical probes to assess the utility of an E. coli Y371H/M173L-GalK-overproducing strain to generate unnatural sugar-1-phosphates in vivo. Remarkably, the in vivo conversion of both unnatural sugars rival that demonstrated in vitro. This notable in vivo success stands as the first step toward constructing short sugar-activation pathways in vivo and, ultimately, in vivo natural-product glycorandomization.     

The cytotoxicity of trifluoromethyl boron derivatives and mode of action in human Tmolt3 T leukemic cells

Trifluoromethylboron derivatives proved to be cytotoxic in a number of murine and human leukemic and lymphoma screens. Solid tumor growth, e.g. glioma HS 683, breast Mck‐7 and colon adenocarcinoma SW480, was reduced significantly by the compounds. Human Tmolt₃ T‐cell leukemia DNA synthesis was inhibited preferentially by the derivatives, with marginal effects on RNA and protein syntheses, after 60 min at 100 µM . The agents appeared to act by multiple mechanisms in that they inhibited the activities of DNA polymerase α, dihydrofolate reductase and nucleosides, significantly within 60 min at 100 µM . Deoxyribonucleotide pools were reduced after 60 min incubation with the compounds. Tmolt₃ DNA fragmentation and reduced ct‐DNA viscosity were evident after 24 h of incubation at 100 µM . ct‐DNA thermal denaturation studies indicated that the agents caused some type of interaction with the bases of DNA. Copyright © 2000 John Wiley & Sons, Ltd.