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Catalytic mechanism of DNA topoisomerase IB

MOLECULAR CELL 2000年第6期5卷 1035-1041页

作者： Krogh, BO Shuman, S Sloan Kettering Inst Program Mol Biol New York NY 10021 USA

Type IB topoisomerases and tyrosine recombinases are structurally homologous strand transferases that act through DNA-(3'-phosphotyrosyl)-enzyme intermediates. A constellation of conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265 in vaccinia topoisomerase) catalyzes transesterification of tyrosine to the scissile phosphodiester. We used 8'-bridging phosphorothiolate-modified DNAs to implicate Lys-167 as a general acid catalyst. The lower pK(a), of the 5'-S leaving group versus 5'-O restored activity to the K167A mutant, whereas there was no positive thio effect for mutants R223A and H265A. The lysine is located atop a flexible hairpin loop, and it shifts into the minor groove upon DNA binding. Coupling of conformational changes in a general acid loop to covalent catalysis of phosphoryl transfer is one of several mechanistic features shared by the topoisomerase/recombinase and protein phosphatase superfamilies.

关键词：氨基酸取代精氨酸/遗传学精氨酸/代谢碱基序列催化作用保守序列/遗传学 DNA/遗传学 DNA/代谢 DNA拓扑异构酶类 Ⅰ型/化学 DNA拓扑异构酶类 Ⅰ型/遗传学 DNA拓扑异构酶类 Ⅰ型/代谢组氨酸/遗传学组氨酸/代谢动力学赖氨酸/遗传学赖氨酸/代谢模型分子分子序列数据突变酪氨酸/代谢痘苗病毒/酶学病毒蛋白质类/化学病毒蛋白质类/遗传学病毒蛋白质类/代谢

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High resolution structure of the phosphohistidine-activated form of Escherichia coli cofactor-dependent phosphoglycerate mutase

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JOURNAL OF BIOLOGICAL CHEMISTRY 2001年第5期276卷 3247-3253页

作者： Bond, CS White, MF Hunter, WN Univ Dundee Wellcome Trust Bioctr Dundee DD1 5EH Scotland

The active conformation of the dimeric cofactor-dependent phosphoglycerate mutase (dPGM) from Escherichia coli has been elucidated by crystallographic methods to a resolution of 1.25 Angstrom (R-factor 0.121;R-free 0.168), The active site residue His(10), central in the catalytic mechanism of dPGM, is present as a phosphohistidine with occupancy of 0.28, The structural changes on histidine phosphorylation highlight various features that are significant in the catalytic mechanism, The C-terminal 10-residue tail, which is not observed in previous dPGM structures, is well ordered and interacts with residues implicated in substrate binding;the displacement of a loop adjacent to the active histidine brings previously overlooked residues into positions where they may directly influence catalysis. E. coli dPGM, like the mammalian dPGMs, is a dimer, whereas previous structural work has concentrated on monomeric and tetrameric yeast forms. We can now analyze the sequence differences that cause this variation of quaternary structure.

关键词：氨基酸序列结合部位大肠杆菌/酶学组氨酸/代谢模型分子分子序列数据磷酸甘油酸酯变位酶/化学磷酸甘油酸酯变位酶/代谢磷酰化蛋白质折叠蛋白质结构四级序列同源性氨基酸

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Evidence for the head domain movement of the Rieske iron-sulfur protein in electron transfer reaction of the cytochrome bc₁ complex

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第11期274卷 7146-7152页

作者： Tian, H White, S Yu, L Yu, CA Oklahoma State Univ Dept Biochem & Mol Biol Stillwater OK 74078 USA

The three-dimensional structure of the mitochondrial cytochrome bc(1) complex suggests that movement of the extramembrane domain (head) of the Rieske iron-sulfur protein (ISP) may play an important role in electron transfer. Such movement requires flexibility in the neck region of ISP, since the head and transmembrane domains of the protein are rather rigid. To test this hypothesis, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with cysteine substitution at various positions in the ISP neck (residues 39-48) were generated and characterized. The mutants with a single cysteine substitution at Ala(42) or Val(44) and a double cysteine substitution at Val(44) and Ala(46) (VQA-CQC) or at Ala(42) and Ala(46) (ADVQA-CDVQC) have photosynthetic growth rates comparable with that of complement cells, Chromatophore membrane and intracytoplasmic membrane (ICM) prepared from these mutants have cytochrome bc(1) complex activity similar to that in the complement membranes, indicating that flexibility of the neck region of ISP was not affected by these cysteine substitutions. Mutants with a double cysteine substitution at Ala(42) and Val(44) (ADV-CDC) or at Pro(40) and Ala(42) (PSA-CSC) have a retarded (50%) or no photosynthetic growth rate, respectively. The ADV-CDC or PSA-CSC mutant ICM contains 20 or 0% of the cytochrome bc(1) complex activity found in the complement ICM, However, activity can be restored by the treatment with beta-mercaptoethanol (beta-ME), The restored activity is diminished upon removal of beta-ME but is retained if the beta-ME-treated membrane is treated with the sulfhydryl reagent N-ethylmaleimide or p-chloromercuribenzoic acid, These results indicate that the loss of bc(1) complex activity in the ADV-CDC or PSA-CSC mutant membranes is due to disulfide bond formation, which increases the rigidity of ISP neck and, in turn, decreases the mobility of the head domain, Using the conditions developed for the isolation of His-tagge

关键词：碱基序列 DNA引物二硫化物类/化学电子自旋共振谱学电子转运电子传递复合物Ⅲ/化学电子传递复合物Ⅲ/遗传学电子传递复合物Ⅲ/代谢组氨酸/化学组氨酸/代谢铁硫蛋白质类/代谢诱变定点蛋白质结合球形红杆菌/酶学球形红杆菌/遗传学

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Heteronuclear NMR and crystallographic studies of wild-type and H187Q Escherichia coli uracil DNA glycosylase:: Electrophilic catalysis of uracil expulsion by a neutral histidine 187

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BIOCHEMISTRY 1999年第37期38卷 11876-11886页

作者： Drohat, AC Xiao, GY Tordova, M Jagadeesh, J Pankiewicz, KW Watanabe, KA Gilliland, GL Stivers, JT Univ Maryland Maryland Biotechnol Inst Ctr Adv Res Biotechnol Rockville MD 20850 USA Natl Inst Stand & Technol Rockville MD 20850 USA Pharmasset Inc Atlanta GA 30345 USA

The nature of the putative general acid His187 in the reaction catalyzed by Escherichia coli uracil DNA glycosylase (UDG) was investigated using X-ray crystallography and NMR spectroscopy. The crystal structures of H187Q UDG, and its complex with uracil, have been solved at 1.40 and 1.60 Angstrom resolution, respectively. The structures are essentially identical to those of the wild-type enzyme, except that the side chain of Gln187 is turned away from the uracil base and cannot interact with uracil O2. This result provides a structural basis for the similar kinetic properties of the H187Q and H187A enzymes. The ionization state of His187 was directly addressed with H-1-N-15 NMR experiments optimized for histidine ring spin systems, which established that His 187 is neutral in the catalytically active state of the enzyme (pK(a) <5.5). These NMR experiments also show that His187 is held in the N-epsilon 2-H tautomeric form, consistent with the crystallographic observation of a 2.9 Angstrom hydrogen bond from the backbone nitrogen of Ser189 to the ring N-delta 1 of His187. The energetic cost of breaking this hydrogen bond may contribute significantly to the low pK(a) of His 187. Thus, the traditional view that a cationic His 187 donates a proton to uracil O2 is incorrect. Rather, we propose a concerted mechanism involving general base catalysis by Asp64 and electrophilic stabilization of the developing enolate on uracil O2 by a neutral His187.

关键词：结合部位/遗传学碳同位素催化作用结晶学 X线 DNA糖基化酶类酶稳定性大肠杆菌/酶学谷氨酰胺/遗传学组氨酸/化学组氨酸/遗传学组氨酸/代谢氢键合氢离子浓度诱变定点 N-糖基水解酶类/化学 N-糖基水解酶类/遗传学 N-糖基水解酶类/代谢氮同位素核磁共振生物分子质子底物特异性尿嘧啶/化学尿嘧啶/代谢尿嘧啶-DNA糖苷酶

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The refined atomic structure of carbonic anhydrase II at 1.05 Å resolution:: implications of chemical rescue of proton transfer

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ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY 2003年第1期59卷 93-104页

作者： Duda, D Govindasamy, L Agbandje-McKenna, M Tu, CK Silverman, DN McKenna, R Univ Florida Dept Biochem & Mol Biol Gainesville FL 32610 USA Univ Florida Dept Pharmacol & Therapeut Gainesville FL 32610 USA

Using synchrotron radiation and a CCD detector, X-ray data have been collected at 100 K for the His64Ala mutant of human carbonic anhydrase II complexed with 4-methylimidazole (4-MI) to a maximal 1.05 Angstrom resolution, allowing full anisotropic least-squares refinement. The refined model has a conventional R factor of 15.7% for all reflections. The C-alpha coordinates of the model presented here have an r.m.s. deviation of 0.10 Angstrom relative to the previously determined structure at 1.6 Angstrom resolution. Several amino-acid residues (six of the 255 observed) have been identified with multiple rotamer side-chain conformations. C, N and O atoms can be differentiated with selective electron-density map contouring. The estimated standard deviations for all main-chain non-H atom bond lengths and angles are 0.013 and 0.030 Angstrom, respectively, based on unrestrained full-matrix least-squares refinement. This structure gives detailed information about the tetrahedrally arranged zinc ion coordinated by three histidine N atoms (His94 N-epsilon2, His96 N-epsilon2 and His119 N-delta1) and a water/hydroxide, the multiple binding sites of the proton chemical rescue molecule 4-MI and the solvent networks linking the zinc-bound water/hydroxide and 4-MI molecules. This structure presents the highest resolution structure of a carbonic anhydrase isozyme so far determined and adds to the understanding of proton-transfer processes.

关键词：氨基酸取代结合部位碳酸酐酶Ⅱ/化学碳酸酐酶Ⅱ/遗传学碳酸酐酶Ⅱ/代谢结晶学 X线/方法回旋加速器组氨酸/遗传学组氨酸/代谢氢氧化物/化学氢氧化物/代谢汞/化学汞/代谢模型分子蛋白质构象质子水/化学锌/代谢人类

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AUTOREGULATION OF GENE-EXPRESSION

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ANNUAL REVIEW OF MICROBIOLOGY 1975年第1期29卷 275-299页

作者： CALHOUN, DH HATFIELD, GW CITY UNIV NEW YORK MT SINAI SCH MED DEPT MICROBIOL NEW YORK NY 10029 USA UNIV CALIF SCH MED DEPT MED MICROBIOL IRVINE CA 92664 USA

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A study of his-tagged alkaline phosphatase immobilization on a nanoporous nickel-titanium dioxide film

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ANALYTICAL BIOCHEMISTRY 2001年第2期292卷 307-310页

作者： Zhang, JK Cass, AEG Univ London Imperial Coll Sci Technol & Med Dept Biochem London SW7 2AY England

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FRACTIONATION OF HUMAN PAROTID SALIVARY PROTEINS AND ISOLATION OF AN HISTIDINE-RICH ACIDIC PEPTIDE WHICH SHOWS HIGH AFFINITY FOR HYDROXYAPATITE SURFACES

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ARCHIVES OF ORAL BIOLOGY 1975年第9期20卷 553-&页

作者： HAY, DI FORSYTH DENT CTR DEPT CHEM140 THE FENWAYBOSTONMA 02115

Anion exchange chromatography of the proteins from litre quantities of parotid saliva has been shown to be a particularly appropriate technique for obtaining substantial amounts of the more acidic macromolecular components present in this secretion. One specific component obtained by this means was further purified by the use of gel permeation chromatography using a recycling *** material was remarkable for its high content of histidine (190 residues1000) and arginine (111 residues1000) and for its elevated levels of the dicarboxylic amino acids (233 residues1000), particularly aspartate (144 residues1000). A value for its minimum molecular weight, 4520, calculated from the amino acid composition, was in good agreement with the value of 4510 obtained by analytical ultracentrifugation. Its isoelectric point was found to be 7.04 at 20 °C and carbohydrate was absent. Interest in this component arises as a consequence of its high affinity for hydroxyapatite and dental enamel surfaces, suggesting a possible role for this material in the early stages of the formation of the acquired enamel pellicle.

关键词：氨基酸类/分析碳水化合物/分析化学分馏法色谱法凝胶色谱法离子交换组氨酸/分离和提纯组氨酸/代谢羟基磷灰石类/代谢等电聚焦分子量腮腺肽类/分离和提纯肽类/代谢蛋白质类/分析唾液/分析青少年成年人女(雌)性人类男(雄)性

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EFFECT OF CHLORAMPHENICOL AND STARVATION FOR AN ESSENTIAL AMINO-ACID ON POLYPEPTIDE AND POLYRIBONUCLEOTIDE SYNTHESIS IN ESCHERICHIA-COLI INFECTED WITH BACTERIOPHAGE-T4

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ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 1975年第2期169卷 406-414页

作者： WITMER, HJ BAROS, A FORBES, J UNIV ILLINOIS DEPT BIOL SCICHICAGOIL 60680

Escherichia coli 3110 (leu − , trpE − , suA + ) and E. coli 2054 (leu − , trpE , suA ) were infected with bacteriophage T4. The effect of chloramphenicol and leucine-depletion on prereplicative transcription was studied. Both conditions resulted in a manyfold reduction in the rate of phage-specific protein synthesis. Chloramphenicol, added 5 min before infection, limited transcription of the viral genome to promoter proximal genes whereas promoter proximal and distal genes were transcribed during leucine-starvation. The size distribution of nascent T4 phage-specific polypeptides was determined by passing extracts of infected cells through columns of G200 Sephadex. Extract proteins were reduced and alkylated prior to chromatography. Polypeptides made in normally infected cells had an average molecular weight of 23,000–26,000. Polypeptides made in leucine-depleted cells had an average molecular weight of 8,000–12,000 compared to an average molecular weight of 1000 for polypeptides made in chloramphenicol-treated hosts. It is proposed that many phage-specific prereplicative transcripts are prematurely terminated in the absence of some minimal amount of concurrent ribosomal movement. These minimal conditions are met during leucine-starvation but not during treatment with chloramphenicol.

关键词：氨基酸类必需/代谢精氨酸/代谢离心法梯密度氯霉素/药理学大肠杆菌噬菌体/药物作用大肠杆菌噬菌体/代谢 DNA病毒/药物作用 DNA病毒/代谢大肠杆菌/药物作用大肠杆菌/代谢组氨酸/代谢亮氨酸/代谢甲硫氨酸/代谢突变肽生物合成蛋白质生物合成/药物作用 RNA 细菌/生物合成 RNA 病毒/生物合成时间因素转录遗传/药物作用色氨酸/代谢缬氨酸/代谢

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A comparison of mutation spectra detected by the Escherichia coli Lac⁺ reversion assay and the Salmonella typhimurium His⁺ reversion assay

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MUTAGENESIS 2000年第4期15卷 317-323页

作者： Ohta, T Watanabe-Akanuma, M Yamagata, H Tokyo Univ Pharm & Life Sci Sch Life Sci Tokyo 1920392 Japan Inst Environm Toxicol Tokyo 1870011 Japan

Each of the Escherichia coli tester strains in the WP3101P-WP3106P series contains an F' plasmid with a different base substitution mutation within the lacZ gene. Each of the six possible base substitution mutations, therefore, can be assayed with these strains by Lac(+) reversion. We used the strains to characterize the mutational profiles of 21 chemical mutagens, including alkylating agents, base analogs and oxidative compounds. We also assayed the mutagens with Salmonella typhimurium tester strains TA7002, TA7004 and TA7005, which detect A . T --> T . A, G . C --> A . T and G . C --> T . A mutations, respectively, and we compared the sensitivity and specificity of the two systems. Escherichia coli strain WP3102P was more sensitive than the *** strains to G . C --> A . T transitions Induced by N-4-aminocytidine, 5-azacytidine, cumene hydroperoxide (CHP), t-butyl hydroperoxide (BHP), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), methyl methane sulfonate and N-ethyl-N-nitrosourea (ENU), while the reverse was true for G . C --> A . T transitions induced by a-aminopurine and phosmet, Escherichia coli strain WP3104P, which detects G . C --> T . A transversions, was superior to the *** strains in detecting transversions induced by N-4-aminocytidine, 5-azacytidine, 5-diazouracil, CHP, BHP, ENNG, ENU, 4-nitroquinoline 1-oxide (4-NQO) and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), Escherichia coli WP3105P was also more sensitive than *** to A . T --> T . A transversions induced by N-methyl-N-nitrosourea (MNU), CHP and 4-NQO, but it was less sensitive to those induced by ENNG, ENU and 2-amino-purine, The present results indicate that the *** Lac(+) reversion system with tester strains WP3101P-WP3106P is as sensitive as the *** His(+) reversion system for the detection of specific mutations induced by a variety of direct mutagens.

关键词： 2-氨基嘌呤/代谢 4-硝基喹啉-1-氧化物/代谢烷化剂/代谢阿扎胞苷/代谢苯衍生物/代谢胞苷/类似物和衍生物胞苷/代谢 DNA/药物作用 DNA突变分析/方法剂量效应关系药物大肠杆菌/遗传学乙基亚硝脲/代谢甲醛/代谢呋喃类/代谢呋喃基糖酰胺/代谢乙二醛/代谢组氨酸/代谢乳糖操纵子/遗传学甲硝基亚硝胍/类似物和衍生物甲硝基亚硝胍/代谢诱变力试验/方法诱变剂氧化剂/代谢亚胺硫磷/代谢质粒/代谢点突变/药物作用鼠伤寒沙门菌/遗传学叠氮化钠/代谢尿嘧啶/类似物和衍生物尿嘧啶/代谢叔丁基氢过氧化物/代谢

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