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N-alkanamines as substrates to probe the hydrophobic region of bovine serum amine oxidase active site:: A kinetic and spectroscopic study

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ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 2007年第1期465卷 50-60页

作者： Di Paolo, Maria Luisa Pesce, Carmine Lunelli, Michele Scarpa, Marina Rigo, Adelio Univ Padua Dept Chim Biol I-35121 Padua Italy Univ Trent Dipartimento Fis I-38050 Trento Italy INBB Rome Italy

Kinetic and spectroscopic studies were carried out to study the role of hydrophobic effect on the activity of bovine serum amine oxidase (BSAO). Increasing the chain length of the substrates (linear aliphatic primary monoamines), the affinity for the active site increases while the catalytic constant decreases in accordance with a relative low value of dielectric constant (about 10) estimated for the micro-environment of BSAO active site using a fluorescent probe sensitive to solvent polarity. The aliphatic chain of 1-aminononane induces a shift in the pK(a), of the product Schiff base, the hydrolysis of which appears to be a rate-determining step of the reaction. Furthermore, circular dichroism studies highlighted the "flexibility" of BSAO secondary structure that can explain the wide substrate specificity of this enzyme. These results should be useful to elucidate the substrate/inhibitor preferences of CuAOs, in particular of the human enzyme. (c) 2007 Elsevier Inc. All rights reserved.

关键词： structure-function relationships enzyme kinetics microenvironment dielectric properties copper-containing amine oxidases fluorescent probes

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Re-engineering cytochrome P450 2B11dH for enhanced metabolism of several substrates including the anti-cancer prodrugs cyclophosphamide and ifosfamide

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ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 2007年第2期458卷 167-174页

作者： Sun, Ling Chen, Chong S. Waxman, David J. Liu, Hong Halpert, James R. Kumar, Santosh Univ Texas Med Branch Dept Pharmacol & Toxicol Galveston TX 77555 USA Boston Univ Dept Biol Boston MA 02215 USA Chinese Acad Sci Shanghai Inst Mat Med Drug Discovery & Design Ctr Shanghai 201203 Peoples R China

Based on recent directed evolution of P450 2B1, six P450 2B11 mutants at three positions were created in an N-terminal modified construct termed P450 2B11 dH and characterized for enzyme catalysis using five substrates. Mutant I209A demonstrated a 3.2-fold enhanced k(cat)/K-m for 7-ethoxy-4-trifluoromethylcourmarin O-deethylation, largely due to a dramatic decrease in K-m (0.72 mu M vs. 18 mu M). 1209A also demonstrated enhanced selectivity for testosterone 16 beta-hydroxylation over 16 alpha-hydroxylation. In contrast, V183L showed a 4-fold increased k(cat) for 7-benzyloxyresorufin debenzylation and a 4.7-fold increased k(cat)/K-m for testosterone 16a-hydroxylation. V183L also displayed a 1.7-fold higher k(cat)/K-m than P450 2B11dH with the anti-cancer prodrugs cyclophosphamide and ifosfamide, resulting from a similar to 4-fold decrease in Km. Introduction of the V183L mutation into full-length P450 21311 did not enhance the k(cat)/K-m. Overall, the re-engineered P450 2B11dH enzymes exhibited enhanced catalytic efficiency with several substrates including the anti-cancer prodrugs. (c) 2007 Elsevier Inc. All rights reserved.

关键词： cytochrome p450 P4502B11 P450 engineering structure-function relationships site-directed mutagenesis enzyme catalysis

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Ligand-selective determinants in gonadotropin receptors

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MOLECULAR AND CELLULAR ENDOCRINOLOGY 2007年第26期260卷 144-152页

作者： Bogerd, J. Univ Utrecht Dept Endocrinol NL-3584 CH Utrecht Netherlands

In mammals, the interactions between gonadotropins and their cognate receptors are highly specific;unintended cross-reactivity under normal physiological conditions has not been observed. This paper summarizes the comparative structure-function studies that aim at elucidating the molecular basis of the ligand selectivity. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

关键词： gonadotropin receptors ligand selectivity structure-function relationships

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The unique pharmacology of the scorpion α-like toxin Lqh3 is associated with its flexible C-tail

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FEBS JOURNAL 2007年第8期274卷 1918-1931页

作者： Karbat, Izhar Kahn, Roy Cohen, Lior Ilan, Nitza Gilles, Nicolas Corzo, Gerardo Froy, Oren Gur, Maya Albrecht, Gudrun Heinemann, Stefan H. Gordon, Dalia Gurevitz, Michael Tel Aviv Univ George S Wise Fac Life Sci Dept Plant Sci IL-69978 Tel Aviv Israel CEA Saclay Dept Ingn Prot F-91191 Gif Sur Yvette France Univ Nacl Autonoma Mexico Inst Biotecnol Cuernavaca 62191 Morelos Mexico Univ Jena Dept Biophys Ctr Mol Biomed D-6900 Jena Germany

The affinity of scorpion alpha-toxins for various voltage-gated sodium channels (Na(v)s) differs considerably despite similar structures and activities. It has been proposed that key bioactive residues of the five-residue-turn (residues 8-12) and the C-tail form the NC domain, whose topology is dictated by a cis or trans peptide-bond conformation between residues 9 and 10, which correlates with the potency on insect or mammalian Na(v)s. We examined this hypothesis using Lqh3, an alpha-like toxin from Leiurus quinquestriatus hebraeus that is highly active in insects and mammalian brain. Lqh3 exhibits slower association kinetics to Na(v)s compared with other alpha-toxins and its binding to insect Na(v)s is pH-dependent. Mutagenesis of Lqh3 revealed a bi-partite bioactive surface, composed of the Core and NC domains, as found in other alpha-toxins. Yet, substitutions at the five-residue turn and stabilization of the 9-10 bond in the cis conformation did not affect the activity. However, substitution of hydrogen-bond donors/acceptors at the NC domain reduced the pH-dependency of toxin binding, while retaining its high potency at Drosophila Na(v)s expressed in Xenopus oocytes. Based on these results and the conformational flexibility and rearrangement of intramolecular hydrogen-bonds at the NC domain, evident from the known solution structure, we suggest that acidic pH or specific mutations at the NC domain favor toxin conformations with high affinity for the receptor by stabilizing the bound toxin-receptor complex. Moreover, the C-tail flexibility may account for the slower association rates and suggests a novel mechanism of dynamic conformer selection during toxin binding, enabling alpha-like toxins to affect a broad range of Na(v)s.

关键词： pH-dependent toxin binding scorpion alpha-like toxin structure-function relationships toxin effect on inactivation toxin receptor site on sodium channel

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structure-function relationships in factor IX and factor IXa

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TRENDS IN CARDIOVASCULAR MEDICINE 2003年第1期13卷 39-45页

作者： Schmidt, AE Bajaj, SP St Louis Univ Hlth Sci Ctr Div Hematol Oncol Dept Pharmacol & Physiol Sci St Louis MO 63110 USA St Louis Univ Hlth Sci Ctr Dept Internal Med St Louis MO 63110 USA

Factor IX (FIX) consists of an N-terminal gamma-carboxyglutamic acid (Gla) domain followed by two epidermal growth factor (EGF)-like domains, and the C-terminal serine protease domain. During physiologic coagulation, one of the activators of FIX is the FVIIa/tissue factor (TF) complex. In this reaction, the Gla and EGF1 domains of FIX are thought to interact with TF. The FIXa that is generated then combines with FVIIIa on the platelet surface to activate FX in the coagulation cascade. In this assembly, the protease domain and possibly the EGF2 domain of FIXa are thought to provide the primary specificity in binding to FVIIIa. Disruption of the interaction of FIX/FX with TF and of the FIXa:FVIIIa interface may provide a pharmacologic target as an alternative strategy for the development of antithrombotic agents. (C) 2003, Elsevier Science Inc.

关键词： Factor IX Factor IXa 因子Ⅸ 因子ⅨA Anticoagulants structure-function relationships Fixed Protease Domain Factor IXa

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Multivariate analysis of conserved sequence-structure relationships in kinesins: Coupling of the active site and a tubulin-binding sub-domain

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JOURNAL OF MOLECULAR BIOLOGY 2007年第5期368卷 1231-1248页

作者： Grant, Barry J. McCammon, J. Andrew Caves, Leo S. D. Cross, Robert A. Univ Calif San Diego Dept Chem & Biochem La Jolla CA 92093 USA Univ Calif San Diego Howard Hughes Med Inst La Jolla CA 92093 USA Univ York Dept Biol York YO10 5YW N Yorkshire England Marie Curie Res Inst Mol Motors Grp Oxted RH8 0TL England

An extensive computational analysis of available sequence and crystal structure data was used to identify functionally important residue interactions within the motor domain of the kinesin molecular motor. Principal component analysis revealed that all current kinesin crystal structures reside in one of two main conformations, which differ at the active site, and in the position of a microtubule-binding sub-domain relative to a rigid central core. This sub-domain consists of secondary structure elements alpha 4-loop12-alpha 5-loop13 and contains a conserved hydrophilic surface patch that may be involved in strong binding to microtubules. A hinge point for the sub-domain motion lies near a conserved glycine at position 292. Statistical coupling analysis revealed a network of co-evolving positions that link this region to the nucleotide-binding site, via a highly conserved histidine in the switch I loop. The data are consistent with a model in which the nucleotide status of the active site shifts kinesin between weak and strong binding conformations via reconfiguration of the identified sub-domain. Our data provide a statistically supported framework for further examination of this and other structure-function relationships in the kinesin family. (C) 2007 Elsevier Ltd. All rights reserved.

关键词： kinesin molecular motors sequence analysis structure analysis structure-function relationships

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structure-function relationships of the initiation complex of HIV-1 reverse transcription:: the case of mutant viruses using tRNA^His as primer

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NUCLEIC ACIDS RESEARCH 2003年第19期31卷 5764-5775页

作者： Rigourd, M Goldschmidt, V Brulé, F Morrow, CD Ehresmann, B Ehresmann, C Marquet, R Univ Strasbourg 1 CNRS UPR 9002 IBMC F-67084 Strasbourg France Univ Alabama Dept Cell Biol Birmingham AL 35294 USA

Reverse transcription of HIV-1 RNA is initiated from the 3' end of a tRNA(3)(Lys) molecule annealed to the primer binding site (PBS). An additional interaction between the anticodon loop of tRNA(3)(Lys) and a viral A-rich loop is required for efficient initiation of reverse transcription of the HIV-1 MAL isolate. In the HIV-1 HXB2 isolate, simultaneous mutations of the PBS and the A-rich loop (mutant His-AC), but not of the PBS alone (mutant His) allows the virus to stably utilize tRNA(His) as primer. However, mutant His-AC selects additional mutations during cell culture, generating successively His-AC-GAC and His-AC-AT-GAC. Here, we wanted to establish direct relationships between the evolution of these mutants in cell culture, their efficiency in initiating reverse transcription and the structure of the primer/template complexes in vitro. The initiation of reverse transcription of His and His-AC RNAs was dramatically reduced. However, His-AC-GAC RNA, which incorporated three adaptative point mutations, was reverse transcribed more efficiently than the wild type RNA. Incorporation of two additional mutations decreased the efficiency of the initiation of reverse transcription, which remained at the wild type level. Structural probing showed that even though both His-AC and His-AC-GAC RNAs can potentially interact with the anticodon loop of tRNA(His), only the latter template formed a stable interaction. Thus, our results showed that the selection of adaptative mutations by HIV-1 mutants utilizing tRNA(His) as primer was initially dictated by the efficiency of the initiation of reverse transcription, which relied on the existence of a stable interaction between the mutated A-rich loop and the anticodon loop of tRNA(His).

关键词： Anticodon loop Initiation complex structure-function relationships Reverse Transcription Cell culture HIV-1 Mutants Histidine-Specific tRNA

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Differentiation of postembryonic skin endothelial cells

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JOURNAL OF INVESTIGATIVE DERMATOLOGY 2007年第12期127卷 2710-2712页

作者： Karasek, Marvin Stanford Univ Dept Dermatol Stanford CA 94305 USA

Specific cell markers for selective isolation and growth of cells from the lymphatic and vascular systems have increased our understanding of the structure function relationships of both systems. Using these markers, a subset of blood vessel endothelial cells (BECs) with the properties of lymphatic endothelial cells (LECs) has been identified in psoriasis and eczema. The differentiation potential of BECs and LECs in vivo and in vitro is reviewed.

关键词： Animals Blood Vessels Cell Differentiation Eczema Endothelial Cells 动物血管细胞分化湿疹炎症淋巴结模型生物学银屑病皮肤皮肤疾病 differentiation potential structure-function relationships cell marker Individualized Instruction Eczema Endothelial Cells Cell Differentiation

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Altered Amino Acid Metabolism in Glutamine Dumper1 Plants

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PLANT SIGNALING & BEHAVIOR 2007年第3期2卷 182-184页

作者： Pratelli, Rejane Pilot, Guillaume Univ Bonn Inst Cellular & Mol Bot Bonn Germany

Amino acid metabolism lies at the crossroad between nitrogen assimilation, carbon fixation and secondary metabolism. Because of this central position in plant metabolism, amino acid metabolism is tightly regulated by numerous factors to match both demand from the organs and availability of reduced carbon and inorganic nitrogen. While the amino acid biosynthesis enzymes have been shown to be regulated at the transcriptional and protein levels, the genes involved in amino acid sensing, signal transduction and regulation have not yet been identified. The overexpression of Glutamine Dumper1 leads to a large increase in the amino acid content of the plant and, as we show here, to insensitivity to externally applied amino acids. This phenotype is reminiscent of that of the pig1-1 mutant proposed to display a deregulated metabolism. These data suggest that GDU1 is involved in the regulation of amino acid metabolism and transport. As published previously, the analysis of deletion mutants proves that GDU1's VIMAG domain is important for the function of the protein. The present data show furthermore that other regions participate to this function.

关键词： amino acid metabolism Arabidopsis thaliana regulation structure-function relationships biosynthesis enzyme feedback regulation

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The Same But Different

The Same But Different

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作者： Mark A. Rould

Isomorphous difference methods allow rapid and detailed visualization of localized changes in macromolecular structures, whether as a result of mutation or the binding of ligands. Practical aspects of isomorphous methods and differential crystallography are presented, particularly in their application to the phasing of new structures by multiple isomorphous replacement and the detection and characterization of ligand binding. Techniques for maintaining isomorphism between crystals to maximize the differential signal are covered, as are the computational steps involved in generating difference electron density maps. Frontier applications such as determining single-site ligand-binding affinities crystallographically, high-throughput screening of combinatorial compound libraries, in crystallo competition assays, and inferring protein function via exogenous ligand-binding screens are discussed. less

关键词： Multiple Isomorphous Replacement High-throughput Screening Statistical Calculation Molecular Dynamics Simulation Molecular Replacement Crystallization Phage Display Genomics heavy-atom derivatives binding affinity protein-ligand interactions competition assay Isomorphous methods difference Fourier MIR phasing high-throughput screen combinatorial libraries pharmaceutical design structure-function relationships

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