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1.
J Mol Biol ; 427(12): 2205-19, 2015 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-25861762

RESUMO

Guanine-rich sequences can, under appropriate conditions, adopt a distinctive, four-stranded, helical fold known as a G-quadruplex. Interest in quadruplex folds has grown in recent years as evidence of their biological relevance has accumulated from both sequence analysis and function-specific assays. The folds are unusually stable and their formation appears to require close management to maintain cell health; regulatory failure correlates with genomic instability and a number of cancer phenotypes. Biologically relevant quadruplex folds are anticipated to form transiently in mRNA and in single-stranded, unwound DNA. To elucidate factors, including bound solvent, that contribute to the stability of RNA quadruplexes, we examine, by X-ray crystallography and small-angle X-ray scattering, the structure of a previously reported tetramolecular quadruplex, UGGGGU stabilized by Sr(2+) ions. Crystal forms of the octameric assembly formed by this sequence exhibit unusually strong diffraction and anomalous signal enabling the construction of reliable models to a resolution of 0.88Å. The solvent structure confirms hydration patterns reported for other nucleic acid helical conformations and provides support for the greater stability of RNA quadruplexes relative to DNA. Novel features detected in the octameric RNA assembly include a new crystal form, evidence of multiple conformations and structural variations in the 3' U tetrad, including one that leads to the formation of a hydrated internal cavity.


Assuntos
Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Estrôncio/metabolismo , Cátions Bivalentes/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Conformação de Ácido Nucleico , Espalhamento a Baixo Ângulo , Solventes
2.
Proteins ; 83(1): 188-94, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25354467

RESUMO

DNA repair is fundamental to genome stability and is found in all three domains of life. However many archaeal species, such as Methanopyrus kandleri, contain only a subset of the eukaryotic nucleotide excision repair (NER) homologs, and those present often contain significant differences compared to their eukaryotic homologs. To clarify the role of the NER XPG-like protein Mk0566 from M. kandleri, its biochemical activity and three-dimensional structure were investigated. Both were found to be more similar to human FEN-1 than human XPG, suggesting a biological role in replication and long-patch base excision repair rather than in NER.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Euryarchaeota/enzimologia , Endonucleases Flap/química , Endonucleases Flap/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , DNA/metabolismo , Clivagem do DNA , Humanos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Homologia Estrutural de Proteína , Especificidade por Substrato
3.
Proteins ; 79(6): 1820-9, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21491491

RESUMO

The crystal structure of an archaeal-type phosphoenolpyruvate carboxylase from Clostridium perfringens has been determined based on X-ray data extending to 3 Å. The asymmetric unit of the structure includes two tetramers (each a dimer-of-dimers) of the enzyme. The precipitant, malonate, employed for the crystallization is itself a weak inhibitor of phosphoenolpyruvate carboxylase and a malonate molecule is seen in the active-site in the crystal structure. The allosteric binding sites for aspartate (an inhibitor) and glucose-6-phosphate (an activator) observed in the Escherichia coli and Zea mays phosphoenolpyruvate carboxylase structures, respectively, are not conserved in the C. perfringens structure. Aspartate inhibits the C. perfringens enzyme competitively with respect to the substrate, Mg(++.) phosphoenolpyruvate. A mechanism for inhibition is proposed based on the structure and sequence comparisons with other archaeal-type phosphoenolpyruvate carboxylases with differing sensitivity to inhibition by aspartate.


Assuntos
Ácido Aspártico/metabolismo , Clostridium perfringens/enzimologia , Fosfoenolpiruvato Carboxilase/química , Archaea/enzimologia , Clostridium perfringens/química , Clostridium perfringens/metabolismo , Cristalografia por Raios X , Escherichia coli/enzimologia , Modelos Moleculares , Fosfoenolpiruvato Carboxilase/metabolismo , Estrutura Quaternária de Proteína , Zea mays/enzimologia
4.
Acta Crystallogr D Biol Crystallogr ; 67(Pt 1): 67-74, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21206063

RESUMO

SgrAI is a type II restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-activation with expansion of DNA-sequence specificity. The three-dimensional crystal structures of SgrAI bound to cleaved primary-site DNA and Mg²(+) and bound to secondary-site DNA with either Mg²(+) or Ca²(+) are presented. All three structures show a conformation of enzyme and DNA similar to the previously determined dimeric structure of SgrAI bound to uncleaved primary-site DNA and Ca²(+) [Dunten et al. (2008), Nucleic Acids Res. 36, 5405-5416], with the exception of the cleaved bond and a slight shifting of the DNA in the SgrAI/cleaved primary-site DNA/Mg²(+) structure. In addition, a new metal ion binding site is located in one of the two active sites in this structure, which is consistent with proposals for the existence of a metal-ion site near the 3'-O leaving group.


Assuntos
Clivagem do DNA , DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Streptomyces griseus/enzimologia , Regulação Alostérica , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Ativação Enzimática , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato
5.
PLoS Biol ; 8(12): e1000554, 2010 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-21151881

RESUMO

SgrAI is a type IIF restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-modulation of cleavage activity and sequence specificity. Previous studies have shown that DNA bound dimers of SgrAI oligomerize into an activated form with higher DNA cleavage rates, although previously determined crystal structures of SgrAI bound to DNA show only the DNA bound dimer. A new crystal structure of the type II restriction endonuclease SgrAI bound to DNA and Ca(2+) is now presented, which shows the close association of two DNA bound SgrAI dimers. This tetrameric form is unlike those of the homologous enzymes Cfr10I and NgoMIV and is formed by the swapping of the amino-terminal 24 amino acid residues. Two mutations predicted to destabilize the swapped form of SgrAI, P27W and P27G, have been made and shown to eliminate both the oligomerization of the DNA bound SgrAI dimers as well as the allosteric stimulation of DNA cleavage by SgrAI. A mechanism involving domain swapping is proposed to explain the unusual allosteric properties of SgrAI via association of the domain swapped tetramer of SgrAI bound to DNA into higher order oligomers.


Assuntos
Cálcio/química , DNA/química , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/química , Streptomyces griseus/enzimologia , Regulação Alostérica , Clivagem do DNA , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Dimerização , Eletroforese em Gel de Poliacrilamida , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína
6.
Bioorg Med Chem Lett ; 20(14): 4215-8, 2010 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-20538456

RESUMO

An analysis of the binding motifs of known HIV-1 non-nucleoside reverse transcriptase inhibitors has led to discovery of novel piperidine-linked aminopyrimidine derivatives with broad activity against wild-type as well as drug-resistant mutant viruses. Notably, the series retains potency against the K103N/Y181C and Y188L mutants, among others. Thus, the N-benzyl compound 5k has a particularly attractive profile. Synthesis and SAR are presented and discussed, as well as crystal structures relating to the binding motifs.


Assuntos
Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Mutação , Pirimidinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Descoberta de Drogas , Farmacorresistência Viral/genética , HIV-1/genética , Modelos Moleculares , Pirimidinas/química , Relação Estrutura-Atividade
7.
J Biol Chem ; 285(29): 22651-7, 2010 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-20463019

RESUMO

Heme is a vital molecule for all life forms with heme being capable of assisting in catalysis, binding ligands, and undergoing redox changes. Heme-related dysfunction can lead to cardiovascular diseases with the oxidation of the heme of soluble guanylyl cyclase (sGC) critically implicated in some of these cardiovascular diseases. sGC, the main nitric oxide (NO) receptor, stimulates second messenger cGMP production, whereas reactive oxygen species are known to scavenge NO and oxidize/inactivate the heme leading to sGC degradation. This vulnerability of NO-heme signaling to oxidative stress led to the discovery of an NO-independent activator of sGC, cinaciguat (BAY 58-2667), which is a candidate drug in clinical trials to treat acute decompensated heart failure. Here, we present crystallographic and mutagenesis data that reveal the mode of action of BAY 58-2667. The 2.3-A resolution structure of BAY 58-2667 bound to a heme NO and oxygen binding domain (H-NOX) from Nostoc homologous to that of sGC reveals that the trifurcated BAY 58-2667 molecule has displaced the heme and acts as a heme mimetic. Carboxylate groups of BAY 58-2667 make interactions similar to the heme-propionate groups, whereas its hydrophobic phenyl ring linker folds up within the heme cavity in a planar-like fashion. BAY 58-2667 binding causes a rotation of the alphaF helix away from the heme pocket, as this helix is normally held in place via the inhibitory His(105)-heme covalent bond. The structure provides insights into how BAY 58-2667 binds and activates sGC to rescue heme-NO dysfunction in cardiovascular diseases.


Assuntos
Benzoatos/química , Guanilato Ciclase/química , Guanilato Ciclase/metabolismo , Heme/química , Mimetismo Molecular , Óxido Nítrico/química , Nostoc/enzimologia , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Cristalografia por Raios X , Ativação Enzimática , Ativadores de Enzimas/química , Modelos Moleculares , Mutagênese , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Guanilil Ciclase Solúvel , Relação Estrutura-Atividade
8.
Bioorg Med Chem Lett ; 20(5): 1693-6, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-20138512

RESUMO

Here we report on the discovery of a series of maleimides which have high potency and good selectivity for GSK-3beta. The incorporation of polar groups afforded compounds with good bioavailability. The most potent compound 34 has an IC(50) of 0.6nM for GSK-3beta, over 100-fold selectivity against a panel of other kinases, and shows efficacy in rat osteoporosis models. The X-ray structure of GSK-3beta protein with 34 bound revealed the binding mode of the template and provided insights for future optimization opportunities.


Assuntos
Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Indóis/química , Maleimidas/química , Inibidores de Proteínas Quinases/química , Administração Oral , Animais , Sítios de Ligação , Cristalografia por Raios X , Modelos Animais de Doenças , Descoberta de Drogas , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Indóis/síntese química , Indóis/farmacocinética , Maleimidas/síntese química , Maleimidas/farmacocinética , Camundongos , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Ratos , Relação Estrutura-Atividade
9.
J Appl Crystallogr ; 43(Pt 5): 1261-1270, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-22184477

RESUMO

For the past five years, the Structural Molecular Biology group at the Stanford Synchrotron Radiation Lightsource (SSRL) has provided general users of the facility with fully remote access to the macromolecular crystallography beamlines. This was made possible by implementing fully automated beamlines with a flexible control system and an intuitive user interface, and by the development of the robust and efficient Stanford automated mounting robotic sample-changing system. The ability to control a synchrotron beamline remotely from the comfort of the home laboratory has set a new paradigm for the collection of high-quality X-ray diffraction data and has fostered new collaborative research, whereby a number of remote users from different institutions can be connected at the same time to the SSRL beamlines. The use of remote access has revolutionized the way in which scientists interact with synchrotron beamlines and collect diffraction data, and has also triggered a shift in the way crystallography students are introduced to synchrotron data collection and trained in the best methods for collecting high-quality data. SSRL provides expert crystallographic and engineering staff, state-of-the-art crystallography beamlines, and a number of accessible tools to facilitate data collection and in-house remote training, and encourages the use of these facilities for education, training, outreach and collaborative research.

10.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 65(Pt 11): 1193-6, 2009 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-19923749

RESUMO

An archaeal-type phosphoenolpyruvate carboxylase (PepcA) from Clostridium perfringens has been expressed in Escherichia coli in a soluble form with an amino-terminal His tag. The recombinant protein is enzymatically active and two crystal forms have been obtained. Complete diffraction data extending to 3.13 angstrom resolution have been measured from a crystal soaked in KAu(CN)(2), using radiation at a wavelength just above the Au L(III) edge. The asymmetric unit contains two tetramers of PepcA.


Assuntos
Proteínas Arqueais/química , Proteínas de Bactérias/química , Clostridium perfringens/enzimologia , Fosfoenolpiruvato Carboxilase/química , Proteínas Arqueais/genética , Proteínas de Bactérias/genética , Cristalização , Cristalografia por Raios X , Isoenzimas/química , Isoenzimas/genética , Dados de Sequência Molecular , Fosfoenolpiruvato Carboxilase/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Difração de Raios X
11.
Acta Crystallogr D Biol Crystallogr ; 65(Pt 4): 393-8, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19307723

RESUMO

Uninterpretable electron-density maps were obtained using either MIRAS phases or MR phases in attempts to determine the structure of the type II restriction endonuclease SgrAI bound to DNA. While neither solution strategy was particularly promising (map correlation coefficients of 0.29 and 0.22 with the final model, respectively, for the MIRAS and MR phases and Phaser Z scores of 4.0 and 4.3 for the rotation and translation searches), phase combination followed by density modification gave a readily interpretable map. MR with a distantly related model located a dimer in the asymmetric unit and provided the correct transformation to use in averaging electron density between SgrAI subunits. MIRAS data sets with low substitution and MR solutions from only distantly related models should not be ignored, as poor-quality starting phases can be significantly improved. The bootstrapping strategy employed to improve the initial MIRAS phases is described.


Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/química , Streptomyces griseus/enzimologia , Algoritmos , Cátions Bivalentes/metabolismo , Cristalização , Cristalografia por Raios X , Modelos Químicos , Modelos Moleculares , Oligodesoxirribonucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica
12.
FEBS J ; 275(23): 5810-9, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19021757

RESUMO

Tyr235 of GTP-dependent phosphoenolpyruvate (PEP) carboxykinase is a fully invariant residue. The aromatic ring of this residue establishes an energetically favorable weak anion-quadrupole interaction with PEP carboxylate. The role of Tyr235 in catalysis was investigated via kinetic analysis of site-directed mutagenesis-derived variants. The Y235F change lowered the apparent K(m) for PEP by about six-fold, raised the apparent K(m) for Mn(2+) by about 70-fold, and decreased oxaloacetate (OAA)-forming activity by about 10-fold. These effects were due to an enhanced anion-quadrupole interaction between the aromatic side chain at position 235, which now lacked a hydroxyl group, and PEP carboxylate, which probably increased the distance between PEP and Mn(2+) and consequently affected the phosphoryl transfer step and overall catalysis. For the Y235A and Y235S changes, an elimination of the favorable edge-on interaction increased the apparent K(m) for PEP by four- and six-fold, respectively, and the apparent K(m) for Mn(2+) by eight- and six-fold, respectively. The pyruvate kinase-like activity, representing the PEP dephosphorylation step of the OAA-forming reaction, was affected by the substitutions in a similar way to the complete reaction. These observations indicate that the aromatic ring of Tyr235 helps to position PEP in the active site and the hydroxyl group allows an optimal PEP-Mn(2+) distance for efficient phosphoryl transfer and overall catalysis. The Y235A and Y235S changes drastically reduced the PEP-forming and OAA decarboxylase activities, probably due to the elimination of the stabilizing interaction between Tyr235 and the respective products, PEP and pyruvate.


Assuntos
Fosfoenolpiruvato Carboxiquinase (GTP)/química , Fosfoenolpiruvato/química , Tirosina/química , Substituição de Aminoácidos , Ânions/química , Carboxiliases/química , Carboxiliases/metabolismo , Catálise , Domínio Catalítico/genética , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Ligação de Hidrogênio , Cinética , Manganês/química , Modelos Moleculares , Ácido Oxaloacético/química , Ácido Oxaloacético/metabolismo , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Piruvato Quinase/química , Piruvato Quinase/metabolismo , Tirosina/metabolismo
13.
Acta Crystallogr D Biol Crystallogr ; 64(Pt 12): 1210-21, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19018097

RESUMO

Complete automation of the macromolecular crystallography experiment has been achieved at SSRL through the combination of robust mechanized experimental hardware and a flexible control system with an intuitive user interface. These highly reliable systems have enabled crystallography experiments to be carried out from the researchers' home institutions and other remote locations while retaining complete control over even the most challenging systems. A breakthrough component of the system, the Stanford Auto-Mounter (SAM), has enabled the efficient mounting of cryocooled samples without human intervention. Taking advantage of this automation, researchers have successfully screened more than 200 000 samples to select the crystals with the best diffraction quality for data collection as well as to determine optimal crystallization and cryocooling conditions. These systems, which have been deployed on all SSRL macromolecular crystallography beamlines and several beamlines worldwide, are used by more than 80 research groups in remote locations, establishing a new paradigm for macromolecular crystallography experimentation.


Assuntos
Cristalografia por Raios X/métodos , Coleta de Dados , Complexos Multiproteicos/química , Robótica , Redes de Comunicação de Computadores , Sistemas Computacionais , Cristalização , Cristalografia por Raios X/instrumentação , Processamento Eletrônico de Dados , Complexos Multiproteicos/análise , Interface Usuário-Computador
14.
Nucleic Acids Res ; 36(16): 5405-16, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18701646

RESUMO

The three-dimensional X-ray crystal structure of the 'rare cutting' type II restriction endonuclease SgrAI bound to cognate DNA is presented. SgrAI forms a dimer bound to one duplex of DNA. Two Ca(2+) bind in the enzyme active site, with one ion at the interface between the protein and DNA, and the second bound distal from the DNA. These sites are differentially occupied by Mn(2+), with strong binding at the protein-DNA interface, but only partial occupancy of the distal site. The DNA remains uncleaved in the structures from crystals grown in the presence of either divalent cation. The structure of the dimer of SgrAI is similar to those of Cfr10I, Bse634I and NgoMIV, however no tetrameric structure of SgrAI is observed. DNA contacts to the central CCGG base pairs of the SgrAI canonical target sequence (CR|CCGGYG, | marks the site of cleavage) are found to be very similar to those in the NgoMIV/DNA structure (target sequence G|CCGGC). Specificity at the degenerate YR base pairs of the SgrAI sequence may occur via indirect readout using DNA distortion. Recognition of the outer GC base pairs occurs through a single contact to the G from an arginine side chain located in a region unique to SgrAI.


Assuntos
DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Regulação Alostérica , Pareamento de Bases , Sítios de Ligação , Cálcio/química , Cristalografia por Raios X , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Manganês/química , Modelos Moleculares , Ligação Proteica
15.
Bioorg Med Chem Lett ; 18(15): 4352-4, 2008 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-18632268

RESUMO

A series of benzyl pyridazinones were evaluated as HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). Several members of this series showed good activity against the wild-type virus and NNRTI-resistant viruses. The binding of inhibitor 5a to HIV-RT was analyzed by surface plasmon resonance spectroscopy. Pharmacokinetic studies of 5a in rat and dog demonstrated that this compound has good oral bioavailability in animal species. The crystal structure of a complex between HIV-RT and inhibitor 4c is also described.


Assuntos
Transcriptase Reversa do HIV/antagonistas & inibidores , Piridazinas , Inibidores da Transcriptase Reversa , Animais , Cães , Farmacorresistência Viral/efeitos dos fármacos , Concentração Inibidora 50 , Estrutura Molecular , Piridazinas/síntese química , Piridazinas/química , Piridazinas/farmacologia , Ratos , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Relação Estrutura-Atividade
16.
Bioorg Med Chem Lett ; 17(14): 3835-9, 2007 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-17532214

RESUMO

New modifications on the C-8 4-aminobenzyl unit of the previously reported 3-alkyl-1,8-dibenzylxanthine inhibitors of cPEPCK are presented. The most active compound reported here is the 5-chloro-1,3-dimethyl-1H-pyrazole-4-sulfonic acid amide derivative 2 with an IC(50) of 0.29+/-0.08 microM. An X-ray analysis of a heteroaromatic sulfonamide is presented showing a new pi-pi interaction.


Assuntos
Citosol/enzimologia , Inibidores Enzimáticos/farmacologia , Fosfoenolpiruvato Carboxiquinase (GTP)/antagonistas & inibidores , Xantinas/farmacologia , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Xantinas/química
17.
Biochemistry ; 45(51): 15392-404, 2006 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-17176061

RESUMO

Phenol hydroxylase (PH) belongs to a family of bacterial multicomponent monooxygenases (BMMs) with carboxylate-bridged diiron active sites. Included are toluene/o-xylene (ToMO) and soluble methane (sMMO) monooxygenase. PH hydroxylates aromatic compounds, but unlike sMMO, it cannot oxidize alkanes despite having a similar dinuclear iron active site. Important for activity is formation of a complex between the hydroxylase and a regulatory protein component. To address how structural features of BMM hydroxylases and their component complexes may facilitate the catalytic mechanism and choice of substrate, we determined X-ray structures of native and SeMet forms of the PH hydroxylase (PHH) in complex with its regulatory protein (PHM) to 2.3 A resolution. PHM binds in a canyon on one side of the (alphabetagamma)2 PHH dimer, contacting alpha-subunit helices A, E, and F approximately 12 A above the diiron core. The structure of the dinuclear iron center in PHH resembles that of mixed-valent MMOH, suggesting an Fe(II)Fe(III) oxidation state. Helix E, which comprises part of the iron-coordinating four-helix bundle, has more pi-helical character than analogous E helices in MMOH and ToMOH lacking a bound regulatory protein. Consequently, conserved active site Thr and Asn residues translocate to the protein surface, and an approximately 6 A pore opens through the four-helix bundle. Of likely functional significance is a specific hydrogen bond formed between this Asn residue and a conserved Ser side chain on PHM. The PHM protein covers a putative docking site on PHH for the PH reductase, which transfers electrons to the PHH diiron center prior to O2 activation, suggesting that the regulatory component may function to block undesired reduction of oxygenated intermediates during the catalytic cycle. A series of hydrophobic cavities through the PHH alpha-subunit, analogous to those in MMOH, may facilitate movement of the substrate to and/or product from the active site pocket. Comparisons between the ToMOH and PHH structures provide insights into their substrate regiospecificities.


Assuntos
Oxigenases de Função Mista/química , Complexos Multienzimáticos/química , Compostos Policíclicos/química , Subunidades Proteicas/química , Pseudomonas/enzimologia , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Transporte de Elétrons , Proteínas Reguladoras de Ferro/química , Oxigenases de Função Mista/metabolismo , Complexos Multienzimáticos/metabolismo , Oxirredução , Oxigenases/química , Compostos Policíclicos/metabolismo , Ligação Proteica , Dobramento de Proteína , Subunidades Proteicas/metabolismo , Especificidade por Substrato , Zinco/química
18.
J Biol Chem ; 280(14): 14105-13, 2005 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-15677479

RESUMO

Glucokinase (GCK) serves as the pancreatic glucose sensor. Heterozygous inactivating GCK mutations cause hyperglycemia, whereas activating mutations cause hypoglycemia. We studied the GCK V62M mutation identified in two families and co-segregating with hyperglycemia to understand how this mutation resulted in reduced function. Structural modeling locates the mutation close to five naturally occurring activating mutations in the allosteric activator site of the enzyme. Recombinant glutathionyl S-transferase-V62M GCK is paradoxically activated rather than inactivated due to a decreased S0.5 for glucose compared with wild type (4.88 versus 7.55 mM). The recently described pharmacological activator (RO0281675) interacts with GCK at this site. V62M GCK does not respond to RO0281675, nor does it respond to the hepatic glucokinase regulatory protein (GKRP). The enzyme is also thermally unstable, but this lability is apparently less pronounced than in the proven instability mutant E300K. Functional and structural analysis of seven amino acid substitutions at residue Val62 has identified a non-linear relationship between activation by the pharmacological activator and the van der Waals interactions energies. Smaller energies allow a hydrophobic interaction between the activator and glucokinase, whereas larger energies prohibit the ligand from fitting into the binding pocket. We conclude that V62M may cause hyperglycemia by a complex defect of GCK regulation involving instability in combination with loss of control by a putative endogenous activator and/or GKRP. This study illustrates that mutations that cause hyperglycemia are not necessarily kinetically inactivating but may exert their effects by other complex mechanisms. Elucidating such mechanisms leads to a deeper understanding of the GCK glucose sensor and the biochemistry of beta-cells and hepatocytes.


Assuntos
Diabetes Mellitus Tipo 2/genética , Glucoquinase/genética , Glucoquinase/metabolismo , Mutação Puntual , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sítios de Ligação , Proteínas de Transporte/metabolismo , Criança , Análise Mutacional de DNA , Ativação Enzimática , Estabilidade Enzimática , Feminino , Glucose/metabolismo , Humanos , Hiperglicemia/genética , Hiperglicemia/metabolismo , Recém-Nascido , Masculino , Modelos Moleculares , Linhagem , Gravidez , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
19.
Bioorg Med Chem Lett ; 14(4): 913-7, 2004 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-15012993

RESUMO

A novel series of oxindole-type inhibitors of CDK2 that have heteroatom substituted alkynyl moieties at their C-4 position is described. These novel 4-alkynyl-substituted inhibitors have superior potency relative to their parent compound in free enzyme and in cell based assays. The crystal structure of CDK2 in complex with one of these analogues was determined and gives insight to their increased potency. The biochemical evaluation of a representative derivative is also described.


Assuntos
Quinases relacionadas a CDC2 e CDC28/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina , DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Humanos , Indóis/química , Modelos Moleculares , Conformação Molecular , Paclitaxel/farmacologia , Relação Estrutura-Atividade
20.
Bioorg Med Chem Lett ; 13(21): 3871-4, 2003 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-14552798

RESUMO

The analysis of the X-ray structures of two xanthine inhibitors bound to PEPCK and a comparison to the X-ray structure of GTP bound to PEPCK are reported. The SAR at N-1, N-7 and developing SAR at C-8 are consistent with information gained from the X-ray structures of compounds 1 and 2 bound to PEPCK. Representative N-3 modifications of compound 2 that led to the discovery of 3-cyclopropylmethyl and its carboxy analogue as optimal N-3 groups are presented.


Assuntos
Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Xantinas/síntese química , Xantinas/farmacologia , Cristalografia por Raios X , Guanosina Trifosfato/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Fosfoenolpiruvato Carboxiquinase (ATP)/efeitos dos fármacos , Relação Estrutura-Atividade
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