Identifying redox-sensitive extra-chloroplastic enzymes by homology modeling

Homology modeling has been used to identify extra-chloroplastic enzymes that contain potential disulfide bonds. All of the higher plant fructose bisphosphatases and mitochondrial citrate synthases that have been tested to date, two glyceraldehyde-3-P dehydrogenases, two enolases and one lactate dehydrogenase, are redox-sensitive and may then be redox-regulated in vivo. Apparently, redox-sensitivity is not limited to the chloroplast.     

Mechanism of light modulation: identification of potential redox-sensitive cysteines distal to catalytic site in light-activated chloroplast enzymes.

Light-dependent reduction of target disulfides on certain chloroplast enzymes results in a change in activity. We have modeled the tertiary structure of four of these enzymes, namely NADP-linked glyceraldehyde-3-P dehydrogenase, NADP-linked malate dehydrogenase, sedoheptulose bisphosphatase, and fructose bisphosphatase. Models are based on x-ray crystal structures from non-plant species. Each of these enzymes consists of two domains connected by a hinge. Modeling suggests that oxidation of two crucial cysteines to cystine would restrict motion around the hinge in the two dehydrogenases and influence the conformation of the active site. The cysteine residues in the two phosphatases are located in a region known to be sensitive to allosteric modifiers and to be involved in mediating structural changes in mammalian and microbial fructose bisphosphatases. Apparently, the same region is involved in covalent modification of phosphatase activity in the chloroplast.     

Predicting the oxidation state of cysteines by multiple sequence alignment

MOTIVATION: Protein sequences found in databanks usually do not report post translational covalent modifications such as the oxidation state of cystein (Cys) residues. Accurate prediction of whether a functionally or structurally important Cys occurs in the oxidized or thiol form would be helpful for molecular biology experiments and structure prediction. RESULTS: A new method is presented for predicting the oxidation state of Cys residues based on multiple sequence alignments and on the observation that Cys tends to occur in the same oxidation state within the same protein. The prediction of the redox state of Cys performs above 82%. The oxidation state of Cys correlates with the cellular location of the given protein within the cell, but the correlation is not perfect (up to 70%). We also perform a statistical analysis of the different redox states of Cys found in secondary structures and buried positions, and of the secondary structures linked by disulfide bonds. The results suggest that the natural borderline lies between the different oxidation states of Cys rather than between the half cystines and cysteins. AVAILABILITY: A web server implementing the prediction method is available at http://guitar.rockefeller.edu/approximately andras/cyspred.html CONTACT: fisera@rockefeller.edu     

Identification of potential redox-sensitive cysteines in cytosolic forms of fructosebisphosphatase and glyceraldehyde-3-phosphate dehydrogenase

Tertiary-structure modeling suggests the occurrence of disulfide bonds in the cytosolic form of fructosebisphosphatase (EC 3.1.3.11) in spinach (Spinacia oleracea L.), sugarbeet (Beta vulgaris L.) and potato (Solanum tuberosum L.). Redox modulation could then control the AMP sensitivity of fructosebisphosphatase in the cytosol, as suggested by the experiments of E. Khayat et al. (1993, Plant Physiol. 101, 57–64). Modeling also reveals two cysteine residues correctly positioned to form a disulfide bond and hence potentially redox-sensitive in the cytosolic glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) from the facultative crassulacean metabolism plant Mesembryanthemum crystallinum L.Abbreviations Cys cysteine - DTT dithiothreitol This work was supported by NSF DCB-9018265 and US Department of Energy, Office of Health and Environmental Research, under contract W-31-109-ENG-38, and by laboratory-directed research and development funding from Argonne National Laboratory. We thank Hans J. Bohnert, University of Arizona, for providing the ice plant seeds, Xiaomu Niu and Paul M. Hasegawa, Purdue University, for providing the salt bush roots, Larry Sykora and staff at the UIC Greenhouse for cultivating the ice plants, and Christie Aljets for assistance with some of the activity determinations.     

Selective inactivation of redox-sensitive mitochondrial enzymes during cardiac reperfusion

Reperfusion of ischemic myocardial tissue results in an increase in mitochondrial free radical production and declines in respiratory activity. The effects of ischemia and reperfusion on the activities of Krebs cycle enzymes, as well as enzymes involved in electron transport, were evaluated to provide insight into whether free radical events are likely to affect enzymatic and mitochondrial function(s). An in vivo rat model was utilized in which ischemia is induced by ligating the left anterior descending coronary artery. Reperfusion, initiated by release of the ligature, resulted in a significant decline in NADH-linked ADP-dependent mitochondrial respiration as assessed in isolated cardiac mitochondria. Assays of respiratory chain complexes revealed reduction in the activities of complex I and, to a lesser extent, complex IV exclusively during reperfusion, with no alterations in the activities of complexes II and III. Moreover, Krebs cycle enzymes alpha-ketoglutarate dehydrogenase and aconitase were susceptible to reperfusion-induced inactivation with no decline in the activities of other Krebs cycle enzymes. The decline in alpha-ketoglutarate dehydrogenase activity during reperfusion was associated with a loss in native lipoic acid on the E2 subunit, suggesting oxidative inactivation. Inhibition of complex I in vitro promotes free radical generation. alpha-Ketoglutarate dehydrogenase and aconitase are uniquely susceptible to in vitro oxidative inactivation. Thus, our results suggest a scenario in which inhibition of complex I promotes free radical production leading to oxidative inactivation of alpha-ketoglutarate dehydrogenase and aconitase.     

Oxidation and structural perturbation of redox-sensitive enzymes in injured skeletal muscle

Molecular events that control skeletal muscle injury and regeneration are poorly understood. However, inflammation associated with oxidative stress is considered a key player in modulating this process. To understand the consequences of oxidative stress associated with muscle injury, inflammation, and regeneration, hind-limb muscles of C57Bl/6J mice were studied after injection of cardiotoxin (CT). Within 1 day post-CT injection, polymorphonuclear neutrophilic leukocyte accumulation was extensive. Compared to baseline, tissue myeloperoxidase (MPO) activity was elevated eight- and fivefold at 1 and 7 days post-CT, respectively. Ubiquitinylated protein was elevated 1 day postinjury and returned to baseline by 21 days. Cysteine residues of creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were irreversibly oxidized within 1 day post-CT injection and were associated with protein conformational changes that fully recovered after 21 days. Importantly, protein structural alterations occurred in conjunction with significant decreases in CK activity at 1, 3, and 7 days post-CT injury. Interestingly, elevations in tissue MPO activity paralleled the time course of conformational changes in CK and GAPDH. In combination, these results demonstrate that muscle proteins in vivo are structurally and functionally altered via the generation of reactive oxygen species produced during inflammatory events after muscle injury and preceding muscle regeneration.     

Tertiary structure of calcineurin B by homology modeling

The crystal structure of the calcium-binding protein calmodulin is used to model the immunologically important calcineurin subunit B. The rough structure is produced by computer-aided homology modeling. Refinement of this using molecular dynamics leads to a suggested structure which appears to satisfy reasonable hydrophilicity and hydrogen-bonding criteria. In the absence of a crystal structure, the model may prove useful in modeling of its interactions with the phosphatase catalytic subunit calcineurin A, and help to explain the calcium modulation of this protein.     

Substrate specificity of plant UDP-dependent glycosyltransferases predicted from crystal structures and homology modeling

Plant family 1 UDP-dependent glycosyltransferases (UGTs) catalyze the glycosylation of a plethora of bioactive natural products. In Arabidopsis thaliana, 120 UGT encoding genes have been identified. The crystal-based 3D structures of four plant UGTs have recently been published. Despite low sequence conservation, the UGTs show a highly conserved secondary and tertiary structure. The sugar acceptor and sugar donor substrates of UGTs are accommodated in the cleft formed between the N- and C-terminal domains. Several regions of the primary sequence contribute to the formation of the substrate binding pocket including structurally conserved domains as well as loop regions differing both with respect to their amino acid sequence and sequence length. In this review we provide a detailed analysis of the available plant UGT crystal structures to reveal structural features determining substrate specificity. The high 3D structural conservation of the plant UGTs render homology modeling an attractive tool for structure elucidation. The accuracy and utility of UGT structures obtained by homology modeling are discussed and quantitative assessments of model quality are performed by modeling of a plant UGT for which the 3D crystal structure is known. We conclude that homology modeling offers a high degree of accuracy. Shortcomings in homology modeling are also apparent with modeling of loop regions remaining as a particularly difficult task.     

Predicting 3D structures of transient protein-protein complexes by homology

The paper reports a homology based approach for predicting the 3D structures of full length hetero protein complexes. We have created a database of templates that includes structures of hetero protein-protein complexes as well as domain-domain structures (), which allowed us to expand the template pool up to 418 two-chain entries (at 40% sequence identity). Two protocols were tested-a protocol based on position specific Blast search (Protocol-I) and a protocol based on structural similarity of monomers (Protocol-II). All possible combinations of two monomers (350,284 pairs) in the ProtCom database were subjected to both protocols to predict if they form complexes. The predictions were benchmarked against the ProtCom database resulting to false-true positives ratios of approximately 5:1 and approximately 7:1 and recovery of 19% and 86%, respectively for protocols I and II. From 350,284 trials Protocol-I made only approximately 500 wrong predictions resulting to 0.5% error. In addition, though it was shown that artificially created domain-domain structures can in principle be good templates for modeling full length protein complexes, more sensitive methods are needed to detect homology relations. The quality of the models was assessed using two different criteria such as interfacial residues and overall RMSD. It was found that there is no correlation between these two measures. In many cases the interface residues were predicted correctly, but the overall RMSD was over 6 A and vice versa.     

Sequence of the peh gene of Erwinia carotovora: homology between Erwinia and plant enzymes

Polygalacturonase (Peh) and other pectolytic enzymes play a crucial role in the maceration of vegetables by soft rot Erwinia spp. We have sequenced the peh gene of Erwinia carotovora subsp. carotovora, and identified its product as a precursor of molecular weight 42,639, and a mature protein of molecular weight 42,200. A putative KdgR-binding site was identified in the region 5' to the peh gene. The Peh protein showed significant homology with Peh from tomato. In addition, we have found homologies between pectin methylesterase and pectate lyase from Erwinia and their counterparts in tomato. These homologies are described, and their significance discussed.     

A robust all-atom model for LCAT generated by homology modeling

LCAT is activated by apoA-I to form cholesteryl ester. We combined two structures, phospholipase A₂ (PLA₂) that hydrolyzes the ester bond at the sn-2 position of oxidized (short) acyl chains of phospholipid, and bacteriophage tubulin PhuZ, as C- and N-terminal templates, respectively, to create a novel homology model for human LCAT. The juxtaposition of multiple structural motifs matching experimental data is compelling evidence for the general correctness of many features of the model: i) The N-terminal 10 residues of the model, required for LCAT activity, extend the hydrophobic binding trough for the sn-2 chain 15–20 Å relative to PLA₂. ii) The topography of the trough places the ester bond of the sn-2 chain less than 5 Å from the hydroxyl of the catalytic nucleophile, S181. iii) A β-hairpin resembling a lipase lid separates S181 from solvent. iv) S181 interacts with three functionally critical residues: E149, that regulates sn-2 chain specificity, and K128 and R147, whose mutations cause LCAT deficiency. Because the model provides a novel explanation for the complicated thermodynamic problem of the transfer of hydrophobic substrates from HDL to the catalytic triad of LCAT, it is an important step toward understanding the antiatherogenic role of HDL in reverse cholesterol transport.     

Using multiple templates to improve quality of homology models in automated homology modeling

When researchers build high-quality models of protein structure from sequence homology, it is today common to use several alternative target-template alignments. Several methods can, at least in theory, utilize information from multiple templates, and many examples of improved model quality have been reported. However, to our knowledge, thus far no study has shown that automatic inclusion of multiple alignments is guaranteed to improve models without artifacts. Here, we have carried out a systematic investigation of the potential of multiple templates to improving homology model quality. We have used test sets consisting of targets from both recent CASP experiments and a larger reference set. In addition to Modeller and Nest, a new method (Pfrag) for multiple template-based modeling is used, based on the segment-matching algorithm from Levitt's SegMod program. Our results show that all programs can produce multi-template models better than any of the single-template models, but a large part of the improvement is simply due to extension of the models. Most of the remaining improved cases were produced by Modeller. The most important factor is the existence of high-quality single-sequence input alignments. Because of the existence of models that are worse than any of the top single-template models, the average model quality does not improve significantly. However, by ranking models with a model quality assessment program such as ProQ, the average quality is improved by approximately 5% in the CASP7 test set.     

Mutagenic activation of xenobiotics by plant enzymes

Genetic evidence has indicated that plants can activate certain xenobiotics to mutagens, but biochemical evidence is as yet scarce. Nevertheless, plant microsomal enzymes and peroxidases have been shown to form reactive intermediates, the best studied examples being 2-aminofluorene, benzo[a]pyrene and pentachlorophenol. The latter two xenobiotics are converted to quinoid derivatives which are, in principle, able to redox cycle and generate active oxygen species. In analogy to results obtained in mammalian systems, covalent binding of reactive intermediates to DNA as well as fragmentation of DNA, are proposed as major mechanisms of action of mutagenic plant metabolites.     

Predicting competitive interactions between pioneer plant species by using plant traits

Abstract. A competitive effect hierarchy for 15 Namaqualand pioneer plant species was established by using the mean mass of the phytometer (Dimorphotheca sinuata) when grown in combination with itself and 14 other species. There were no clear groupings of species in the hierarchy. This competitive hierarchy (gradient) indicated which species are strong competitors (resulting in a low phytometer mass) with D. sinuata and which species are weak competitors (resulting in a high phytometer mass). Each plant species has a certain combination of plant traits which determines its life history strategy and competitive ability. Regressions of various plant traits (measured on plants grown singly) against phytometer biomass indicated which traits were significantly correlated. The traits, most being size-related, were: maximum shoot mass, total mass, stem mass, reproductive mass, leaf area, stem allocation, specific leaf area (SLA), vegetative height × diameter, leaf area ratio (LAR); and mean number of days to flower initiation. A forward stepwise multiple regression of the significant traits was used to determine an equation to predict competitive effect.     

Genome modifications in plant cells by custom-made restriction enzymes

Genome editing, i.e. the ability to mutagenize, insert, delete and replace sequences, in living cells is a powerful and highly desirable method that could potentially revolutionize plant basic research and applied biotechnology. Indeed, various research groups from academia and industry are in a race to devise methods and develop tools that will enable not only site-specific mutagenesis but also controlled foreign DNA integration and replacement of native and transgene sequences by foreign DNA, in living plant cells. In recent years, much of the progress seen in gene targeting in plant cells has been attributed to the development of zinc finger nucleases and other novel restriction enzymes for use as molecular DNA scissors. The induction of double-strand breaks at specific genomic locations by zinc finger nucleases and other novel restriction enzymes results in a wide variety of genetic changes, which range from gene addition to the replacement, deletion and site-specific mutagenesis of endogenous and heterologous genes in living plant cells. In this review, we discuss the principles and tools for restriction enzyme-mediated gene targeting in plant cells, as well as their current and prospective use for gene targeting in model and crop plants.     

Sequence annotation of nuclear receptor ligand-binding domains by automated homology modeling

The quality of three-dimensional homology models derived from protein sequences provides an independent measure of the suitability of a protein sequence for a certain fold. We have used automated homology modeling and model assessment tools to identify putative nuclear hormone receptor ligand-binding domains in the genome of Caenorhabditis elegans. Our results indicate that the availability of multiple crystal structures is crucial to obtaining useful models in this receptor family. The majority of annotated mammalian nuclear hormone receptors could be assigned to a ligand-binding domain fold by using the best model derived from any of four template structures. This strategy also assigned the ligand-binding domain fold to a number of C.elegans. sequences without prior annotation. Interestingly, the retinoic acid receptor crystal structure contributed most to the number of sequences that could be assigned to a ligand-binding domain fold. Several causes for this can be suggested, including the high quality of this protein structure in terms of our assessment tools, similarity between the biological function or ligand of this receptor and the modeled genes and gene duplication in C.elegans.     

Building a three-dimensional model of rat albumin molecule by homology modeling

Albumin is known as being able to cleave ester bonds in organophosphates (OP) and serve as a major means of OP detoxification due to its large amount in the blood serum. Most of in vivo toxicological studies are conducted on rodents, mice and rats. To adequately extrapolate results of ligand/albumin interaction studies to a human organism, it is necessary to carry out a comparative analysis of the rat (RSA) and human (HSA) albumin structure by in silico methods. X-ray diffraction analysis of RSA has not been done as yet, and the RSA structure is still undetermined. The aim of this study was to build a three-dimensional (3D) model of RSA by homology modeling. As templates, 14 RSA homologs were selected from the Protein Data Bank. A 3D model of the RSA molecule was built using the RSA primary sequence and 3D models of the selected templates. The 3D model geometry was improved by the energy minimization method. The quality of the model was evaluated by 9 parameters. According to this evaluation, in 97% of protein structures from the comparative dataset these parameters were worse than in the obtained model. It was concluded that this model is suitable for further research, specifically, for in silico studies of the RSA/OP interaction and comparative evolutionary analysis of RSA and HSA.