Bound Substrate (bound + substrate)

Distribution by Scientific Domains


Selected Abstracts


The allene oxide cyclase family of Arabidopsis thaliana , localization and cyclization

FEBS JOURNAL, Issue 10 2008
Florian Schaller
Jasmonates are derived from oxygenated fatty acids (oxylipins) via the octadecanoid pathway and are characterized by a pentacyclic ring structure. They have regulatory functions as signaling molecules in plant development and adaptation to environmental stress. Recently, we solved the structure of allene oxide cyclase 2 (AOC2) of Arabidopsis thaliana, which is, together with the other three AOCs, a key enzyme in the biosynthesis of jasmonates, in that it releases the first cyclic and biologically active metabolite , 12-oxo-phytodienoic acid (OPDA). On the basis of models for the bound substrate, 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid, and the product, OPDA, we proposed that a conserved Glu promotes the reaction by anchimeric assistance. According to this hypothesis, the transition state with a pentadienyl carbocation and an oxyanion is stabilized by a strongly bound water molecule and favorable ,,, interactions with aromatic residues in the cavity. Stereoselectivity results from steric restrictions to the necessary substrate isomerizations imposed by the protein environment. Here, site-directed mutagenesis was used to explore and verify the proposed reaction mechanism. In a comparative analysis of the AOC family from A. thaliana involving enzymatic characterization, in vitro import, and transient expression of AOC,enhanced green fluorescent protein fusion proteins for analysis of subcellular targeting, we demonstrate that all four AOC isoenzymes may contribute to jasmonate biosynthesis, as they are all located in chloroplasts and, in concert with the allene oxide synthase, they are all able to convert 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid into enantiomerically pure cis(+)-OPDA. [source]


Molecular determinants of ligand specificity in family 11 carbohydrate binding modules , an NMR, X-ray crystallography and computational chemistry approach

FEBS JOURNAL, Issue 10 2008
Aldino Viegas
The direct conversion of plant cell wall polysaccharides into soluble sugars is one of the most important reactions on earth, and is performed by certain microorganisms such as Clostridium thermocellum (Ct). These organisms produce extracellular multi-subunit complexes (i.e. cellulosomes) comprising a consortium of enzymes, which contain noncatalytic carbohydrate-binding modules (CBM) that increase the activity of the catalytic module. In the present study, we describe a combined approach by X-ray crystallography, NMR and computational chemistry that aimed to gain further insight into the binding mode of different carbohydrates (cellobiose, cellotetraose and cellohexaose) to the binding pocket of the family 11 CBM. The crystal structure of C. thermocellum CBM11 has been resolved to 1.98 Å in the apo form. Since the structure with a bound substrate could not be obtained, computational studies with cellobiose, cellotetraose and cellohexaose were carried out to determine the molecular recognition of glucose polymers by CtCBM11. These studies revealed a specificity area at the CtCBM11 binding cleft, which is lined with several aspartate residues. In addition, a cluster of aromatic residues was found to be important for guiding and packing of the polysaccharide. The binding cleft of CtCBM11 interacts more strongly with the central glucose units of cellotetraose and cellohexaose, mainly through interactions with the sugar units at positions 2 and 6. This model of binding is supported by saturation transfer difference NMR experiments and linebroadening NMR studies. [source]


Mapping of the active site of glutamate carboxypeptidase II by site-directed mutagenesis

FEBS JOURNAL, Issue 18 2007
Petra Ml, ochová
Human glutamate carboxypeptidase II [GCPII (EC 3.4.17.21)] is recognized as a promising pharmacological target for the treatment and imaging of various pathologies, including neurological disorders and prostate cancer. Recently reported crystal structures of GCPII provide structural insight into the organization of the substrate binding cavity and highlight residues implicated in substrate/inhibitor binding in the S1, site of the enzyme. To complement and extend the structural studies, we constructed a model of GCPII in complex with its substrate, N -acetyl- l -aspartyl- l -glutamate, which enabled us to predict additional amino acid residues interacting with the bound substrate, and used site-directed mutagenesis to assess the contribution of individual residues for substrate/inhibitor binding and enzymatic activity of GCPII. We prepared and characterized 12 GCPII mutants targeting the amino acids in the vicinity of substrate/inhibitor binding pockets. The experimental results, together with the molecular modeling, suggest that the amino acid residues delineating the S1, pocket of the enzyme (namely Arg210) contribute primarily to the high affinity binding of GCPII substrates/inhibitors, whereas the residues forming the S1 pocket might be more important for the ,fine-tuning' of GCPII substrate specificity. [source]


Two alternative modes for optimizing nylon-6 byproduct hydrolytic activity from a carboxylesterase with a ,-lactamase fold: X-ray crystallographic analysis of directly evolved 6-aminohexanoate-dimer hydrolase

PROTEIN SCIENCE, Issue 8 2009
Taku Ohki
Abstract Promiscuous 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity originally obtained in a carboxylesterase with a ,-lactamase fold was enhanced about 80-fold by directed evolution using error-prone PCR and DNA shuffling. Kinetic studies of the mutant enzyme (Hyb-S4M94) demonstrated that the enzyme had acquired an increased affinity (Km = 15 mM) and turnover (kcat = 3.1 s,1) for Ald, and that a catalytic center suitable for nylon-6 byproduct hydrolysis had been generated. Construction of various mutant enzymes revealed that the enhanced activity in the newly evolved enzyme is due to the substitutions R187S/F264C/D370Y. Crystal structures of Hyb-S4M94 with bound substrate suggested that catalytic function for Ald was improved by hydrogen-bonding/hydrophobic interactions between the AldCOOH and Tyr370, a hydrogen-bonding network from Ser187 to AldNH, and interaction between AldNH and Gln27-O, derived from another subunit in the homo-dimeric structure. In wild-type Ald-hydrolase (NylB), Ald-hydrolytic activity is thought to be optimized by the substitutions G181D/H266N, which improve an electrostatic interaction with AldNH (Kawashima et al., FEBS J 2009; 276:2547,2556). We propose here that there exist at least two alternative modes for optimizing the Ald-hydrolytic activity of a carboxylesterase with a ,-lactamase fold. [source]


All three Ca2+ -binding loops of photoproteins bind calcium ions: The crystal structures of calcium-loaded apo-aequorin and apo-obelin

PROTEIN SCIENCE, Issue 3 2005
Lu Deng
HLH, helix,loop,helix; HSQC, heteronuclear single quantum coherence; RMSD, root mean square deviation; SAD, single wavelength anomalous dispersion Abstract The crystal structures of calcium-loaded apoaequorin and apo-obelin have been determined at resolutions 1.7 Å and 2.2 Å, respectively. A calcium ion is observed in each of the three EF-hand loops that have the canonical calcium-binding sequence, and each is coordinated in the characteristic pentagonal bipyramidal configuration. The calcium-loaded apo-proteins retain the same compact scaffold and overall fold as the unreacted photoproteins containing the bound substrate, 2-hydroperoxycoelenterazine, and also the same as the Ca2+ -discharged obelin bound with the product, coelenteramide. Nevertheless, there are easily discerned shifts in both helix and loop regions, and the shifts are not the same between the two proteins. It is suggested that these subtle shifts are the basis of the ability of these photoproteins to sense Ca2+ concentration transients and to produce their bioluminescence response on the millisecond timescale. A mechanism of intrastructural transmission of the calcium signal is proposed. [source]


Structure,function studies of glutamate synthases: A class of self-regulated iron-sulfur flavoenzymes essential for nitrogen assimilation

IUBMB LIFE, Issue 5 2008
Maria Antonietta Vanoni
Abstract Glutamate synthases play with glutamine synthetase an essential role in nitrogen assimilation processes in microorganisms, plants, and lower animals by catalyzing the net synthesis of one molecule of L -glutamate from L -glutamine and 2-oxoglutarate. They exhibit a modular architecture with a common subunit or region, which is responsible for the L -glutamine-dependent glutamate synthesis from 2-oxoglutarate. Here, a PurF- (Type II- or Ntn-) type amidotransferase domain is coupled to the synthase domain, a (,/,)8 barrel containing FMN and one [3Fe-4S]0,+1 cluster, through a ,30 Å-long intramolecular tunnel for the transfer of ammonia between the sites. In bacterial and eukaryotic GltS, reducing equivalents are provided by reduced pyridine nucleotides thanks to the stable association with a second subunit or region, which acts as a FAD-dependent NAD(P)H oxidoreductase and is responsible for the formation of the two low potential [4Fe-4S]+1,+2 clusters of the enzyme. In photosynthetic cells, reduced ferredoxin is the physiological reductant. This review focus on the mechanism of cross-activation of the synthase and glutaminase reactions in response to the bound substrates and the redox state of the enzyme cofactors, as well as on recent information on the structure of the ,, protomer of the NADPH-dependent enzyme, which sheds light on the intramolecular electron transfer pathway between the flavin cofactors. © 2008 IUBMB IUBMB Life, 60(5): 287,300, 2008 [source]


Modified Microperoxidases Exhibit Different Reactivity Towards Phenolic Substrates

CHEMBIOCHEM, Issue 12 2004
Corrado Dallacosta Dr.
Abstract The reactivity of several microperoxidase derivatives with different distal-site environments has been studied. The distal-site environments of these heme peptides include a positively charged one, an uncharged environment, two bulky and doubly or triply positively charged ones, and one containing aromatic apolar residues. The reactivity in the catalytic oxidation of two representative phenols, carrying opposite charges, by hydrogen peroxide has been investigated. This allows the determination of the binding constants and of the electron-transfer rate from the phenol to the catalyst in the substrate/microperoxidase complex. The electron-transfer rates scarcely depend on the redox and charge properties of the phenol, but depend strongly on the microperoxidase. Information on the disposition of the substrate in the adducts with the microperoxidases has been obtained through determination of the paramagnetic contribution to the1H NMR relaxation rates of the protons of the bound substrates. The data show that the electron-transfer rate drops when the substrate binds too far away from the iron and that the phenols bind to microperoxidases at similar distances to those observed with peroxidases. While the reaction rate of microperoxidases with peroxide is significantly smaller than that of the enzymes, the efficiency in the one-electron oxidation of phenolic substrates is almost comparable. Interestingly, the oxyferryl form of the triply positively charged microperoxidases shows a reactivity larger than that exhibited by horseradish peroxidase. [source]