Clustering under the line graph transformation: application to reaction network

Background  

Many real networks can be understood as two complementary networks with two kind of nodes. This is the case of metabolic networks where the first network has chemical compounds as nodes and the second one has nodes as reactions. In general, the second network may be related to the first one by a technique called line graph transformation (i.e., edges in an initial network are transformed into nodes). Recently, the main topological properties of the metabolic networks have been properly described by means of a hierarchical model. While the chemical compound network has been classified as hierarchical network, a detailed study of the chemical reaction network had not been carried out.     

Identification of CDK2 substrates in human cell lysates

Background  

Protein phosphorylation regulates a multitude of biological processes. However, the large number of protein kinases and their substrates generates an enormously complex phosphoproteome. The cyclin-dependent kinases - the CDKs - comprise a class of enzymes that regulate cell cycle progression and play important roles in tumorigenesis. However, despite intense study, only a limited number of mammalian CDK substrates are known. A comprehensive understanding of CDK function requires the identification of their substrate network.     

A versatile polyacrylamide gel electrophoresis based sulfotransferase assay

Background  

Sulfotransferases are a large group of enzymes that regulate the biological activity or availability of a wide spectrum of substrates through sulfation with the sulfur donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS). These enzymes are known to be difficult to assay. A convenient assay is needed in order to better understand these enzymes.     

Predicting functional associations from metabolism using bi-partite network algorithms

Background  

Metabolic reconstructions contain detailed information about metabolic enzymes and their reactants and products. These networks can be used to infer functional associations between metabolic enzymes. Many methods are based on the number of metabolites shared by two enzymes, or the shortest path between two enzymes. Metabolite sharing can miss associations between non-consecutive enzymes in a serial pathway, and shortest-path algorithms are sensitive to high-degree metabolites such as water and ATP that create connections between enzymes with little functional similarity.     

Total sequence decomposition distinguishes functional modules, "molegos" in apurinic/apyrimidinic endonucleases

Background  

Total sequence decomposition, using the web-based MASIA tool, identifies areas of conservation in aligned protein sequences. By structurally annotating these motifs, the sequence can be parsed into individual building blocks, molecular legos ("molegos"), that can eventually be related to function. Here, the approach is applied to the apurinic/apyrimidinic endonuclease (APE) DNA repair proteins, essential enzymes that have been highly conserved throughout evolution. The APEs, DNase-1 and inositol 5'-polyphosphate phosphatases (IPP) form a superfamily that catalyze metal ion based phosphorolysis, but recognize different substrates.     

Bringing metabolic networks to life: convenience rate law and thermodynamic constraints

Background  

Translating a known metabolic network into a dynamic model requires rate laws for all chemical reactions. The mathematical expressions depend on the underlying enzymatic mechanism; they can become quite involved and may contain a large number of parameters. Rate laws and enzyme parameters are still unknown for most enzymes.     

Evolution of a subtilisin-like protease gene family in the grass endophytic fungus Epichlo? festucae

Background  

Subtilisin-like proteases (SLPs) form a superfamily of enzymes that act to degrade protein substrates. In fungi, SLPs can play either a general nutritive role, or may play specific roles in cell metabolism, or as pathogenicity or virulence factors.     

Structural correlations in bacterial metabolic networks

Background  

Evolution of metabolism occurs through the acquisition and loss of genes whose products acts as enzymes in metabolic reactions, and from a presumably simple primordial metabolism the organisms living today have evolved complex and highly variable metabolisms. We have studied this phenomenon by comparing the metabolic networks of 134 bacterial species with known phylogenetic relationships, and by studying a neutral model of metabolic network evolution.     

Molecular cloning,characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01

Aims

To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01.

Methods and Results

The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0·1 mmol l^?1 isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel–nitrilotriacetic acid (Ni²⁺‐NTA) agarose column and their activities characterized. BSH A hydrolysed tauro‐conjugated bile salts optimally at pH 5·0 and 55°C, whereas BSH C hydrolysed glyco‐conjugated bile salts optimally at pH 5·0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety.

Conclusions

BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate‐binding sites, these remain functional through motif conservation.

Significance and Impact of the Study

This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco‐conjugated or tauro‐conjugated bile salts. Future structural homology studies and site‐directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes.     

Functional classification of proteins based on projection of amino acid sequences: application for prediction of protein kinase substrates

Background  

The knowledge about proteins with specific interaction capacity to the protein partners is very important for the modeling of cell signaling networks. However, the experimentally-derived data are sufficiently not complete for the reconstruction of signaling pathways. This problem can be solved by the network enrichment with predicted protein interactions. The previously published in silico method PAAS was applied for prediction of interactions between protein kinases and their substrates.     

Analysis of optimal phenotypic space using elementary modes as applied to Corynebacterium glutamicum

Background  

Quantification of the metabolic network of an organism offers insights into possible ways of developing mutant strain for better productivity of an extracellular metabolite. The first step in this quantification is the enumeration of stoichiometries of all reactions occurring in a metabolic network. The structural details of the network in combination with experimentally observed accumulation rates of external metabolites can yield flux distribution at steady state. One such methodology for quantification is the use of elementary modes, which are minimal set of enzymes connecting external metabolites. Here, we have used a linear objective function subject to elementary modes as constraint to determine the fluxes in the metabolic network of Corynebacterium glutamicum. The feasible phenotypic space was evaluated at various combinations of oxygen and ammonia uptake rates.     

Impact of environmental inputs on reverse-engineering approach to network structures

Background  

Uncovering complex network structures from a biological system is one of the main topic in system biology. The network structures can be inferred by the dynamical Bayesian network or Granger causality, but neither techniques have seriously taken into account the impact of environmental inputs.     

Network analysis of metabolic enzyme evolution in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

The two most common models for the evolution of metabolism are the patchwork evolution model, where enzymes are thought to diverge from broad to narrow substrate specificity, and the retrograde evolution model, according to which enzymes evolve in response to substrate depletion. Analysis of the distribution of homologous enzyme pairs in the metabolic network can shed light on the respective importance of the two models. We here investigate the evolution of the metabolism in E. coli viewed as a single network using EcoCyc.     

Acyl chains of phospholipase d transphosphatidylation products in Arabidopsis cells: a study using multiple reaction monitoring mass spectrometry

Background

Phospholipases D (PLD) are major components of signalling pathways in plant responses to some stresses and hormones. The product of PLD activity is phosphatidic acid (PA). PAs with different acyl chains do not have the same protein targets, so to understand the signalling role of PLD it is essential to analyze the composition of its PA products in the presence and absence of an elicitor.

Methodology/Principal findings

Potential PLD substrates and products were studied in Arabidopsis thaliana suspension cells treated with or without the hormone salicylic acid (SA). As PA can be produced by enzymes other than PLD, we analyzed phosphatidylbutanol (PBut), which is specifically produced by PLD in the presence of n-butanol. The acyl chain compositions of PBut and the major glycerophospholipids were determined by multiple reaction monitoring (MRM) mass spectrometry. PBut profiles of untreated cells or cells treated with SA show an over-representation of 160/18∶2- and 16∶0/18∶3-species compared to those of phosphatidylcholine and phosphatidylethanolamine either from bulk lipid extracts or from purified membrane fractions. When microsomal PLDs were used in in vitro assays, the resulting PBut profile matched exactly that of the substrate provided. Therefore there is a mismatch between the acyl chain compositions of putative substrates and the in vivo products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates.

Conclusions

MRM mass spectrometry is a reliable technique to analyze PLD products. Our results suggest that PLD action in response to SA is not due to the production of a stress-specific molecular species, but that the level of PLD products per se is important. The over-representation of 160/18∶2- and 16∶0/18∶3-species in PLD products when compared to putative substrates might be related to a regulatory role of the heterogeneous distribution of glycerophospholipids in membrane sub-domains.     

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Background  

The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes.     

Human Lsg1 defines a family of essential GTPases that correlates with the evolution of compartmentalization

Background  

Compartmentalization is a key feature of eukaryotic cells, but its evolution remains poorly understood. GTPases are the oldest enzymes that use nucleotides as substrates and they participate in a wide range of cellular processes. Therefore, they are ideal tools for comparative genomic studies aimed at understanding how aspects of biological complexity such as cellular compartmentalization evolved.     

Analysis of proteins with the 'hot dog' fold: Prediction of function and identification of catalytic residues of hypothetical proteins

Background  

The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites.     

A H NMR study of the specificity of α-l-arabinofuranosidases on natural and unnatural substrates

Background

The detailed characterization of arabinoxylan-active enzymes, such as double-substituted xylan arabinofuranosidase activity, is still a challenging topic. Ad hoc chromogenic substrates are useful tools and can reveal subtle differences in enzymatic behavior. In this study, enzyme selectivity on natural substrates has been compared with enzyme selectivity towards aryl-glycosides. This has proven to be a suitable approach to understand how artificial substrates can be used to characterize arabinoxylan-active α-l-arabinofuranosidases (Abfs).

Methods

Real-time NMR using a range of artificial chromogenic, synthetic pseudo-natural and natural substrates was employed to determine the hydrolytic abilities and specificity of different Abfs.

Results

The way in which synthetic di-arabinofuranosylated substrates are hydrolyzed by Abfs mirrors the behavior of enzymes on natural arabinoxylo-oligosaccharide (AXOS). Family GH43 Abfs that are strictly specific for mono-substituted d-xylosyl moieties (AXH-m) do not hydrolyze synthetic di-arabinofuranosylated substrates, while those specific for di-substituted moieties (AXH-d) remove a single l-arabinofuranosyl (l-Araf) group. GH51 Abfs, which are supposedly AXH-m enzymes, can release l-Araf from disubstituted d-xylosyl moieties, when these are non-reducing terminal groups.

Conclusions and general significance

The present study reveals that although the activity of Abfs on artificial substrates can be quite different from that displayed on natural substrates, enzyme specificity is well conserved. This implies that carefully chosen artificial substrates bearing di-arabinofuranosyl d-xylosyl moieties are convenient tools to probe selectivity in new Abfs. Moreover, this study has further clarified the relative promiscuity of GH51 Abfs, which can apparently hydrolyze terminal disubstitutions in AXOS, albeit less efficiently than mono-substituted motifs.     

Genes encoding hub and bottleneck enzymes of the Arabidopsis metabolic network preferentially retain homeologs through whole genome duplication

Background

Whole genome duplication (WGD) occurs widely in angiosperm evolution. It raises the intriguing question of how interacting networks of genes cope with this dramatic evolutionary event.

Results

In study of the Arabidopsis metabolic network, we assigned each enzyme (node) with topological centralities (in-degree, out-degree and between-ness) to measure quantitatively their centralities in the network. The Arabidopsis metabolic network is highly modular and separated into 11 interconnected modules, which correspond well to the functional metabolic pathways. The enzymes with higher in-out degree and between-ness (defined as hub and bottleneck enzymes, respectively) tend to be more conserved and preferentially retain homeologs after WGD. Moreover, the simultaneous retention of homeologs encoding enzymes which catalyze consecutive steps in a pathway is highly favored and easily achieved, and enzyme-enzyme interactions contribute to the retention of one-third of WGD enzymes.

Conclusions

Our analyses indicate that the hub and bottleneck enzymes of metabolic network obtain great benefits from WGD, and this event grants clear evolutionary advantages in adaptation to different environments.     

Molecular evolution and the role of oxidative stress in the expansion and functional diversification of cytosolic glutathione transferases

Background  

Cytosolic glutathione transferases (cGST) are a large group of ubiquitous enzymes involved in detoxification and are well known for their undesired side effects during chemotherapy. In this work we have performed thorough phylogenetic analyses to understand the various aspects of the evolution and functional diversification of cGSTs. Furthermore, we assessed plausible correlations between gene duplication and substrate specificity of gene paralogs in humans and selected species, notably in mammalian enzymes and their natural substrates.