ESTHER, the database of the α/β-hydrolase fold superfamily of proteins: tools to explore diversity of functions

The ESTHER database, which is freely available via a web server (http://bioweb.ensam.inra.fr/esther) and is widely used, is dedicated to proteins with an a/b-hydrolase fold, and it currently contains >30 000 manually curated proteins. Herein, we report those substantial changes towards improvement that we have made to improve ESTHER during the past 8 years since our 2004 update. In particular, we generated 87 new families and increased the coverage of the UniProt Knowledgebase (UniProtKB). We also renewed the ESTHER website and added new visualization tools, such as the Overall Table and the Family Tree. We also address two topics of particular interest to the ESTHER users. First, we explain how the different enzyme classifications (bacterial lipases, peptidases,carboxylesterases) used by different communities of users are combined in ESTHER. Second, we discuss how variations of core architecture or in predicted active site residues result in a more precise clustering of families, and whether this strategy provides trustable hints to identify enzymelike proteins with no catalytic activity

Structural and functional insights into Mimivirus ORFans

<p>Abstract</p> <p>Background</p> <p>Mimivirus isolated from A. <it>polyphaga </it>is the largest virus discovered so far. It is unique among all the viruses in having genes related to translation, DNA repair and replication which bear close homology to eukaryotic genes. Nevertheless, only a small fraction of the proteins (33%) encoded in this genome has been assigned a function. Furthermore, a large fraction of the unassigned protein sequences bear no sequence similarity to proteins from other genomes. These sequences are referred to as ORFans. Because of their lack of sequence similarity to other proteins, they can not be assigned putative functions using standard sequence comparison methods. As part of our genome-wide computational efforts aimed at characterizing Mimivirus ORFans, we have applied fold-recognition methods to predict the structure of these ORFans and further functions were derived based on conservation of functionally important residues in sequence-template alignments.</p> <p>Results</p> <p>Using fold recognition, we have identified highly confident computational 3D structural assignments for 21 Mimivirus ORFans. In addition, highly confident functional predictions for 6 of these ORFans were derived by analyzing the conservation of functional motifs between the predicted structures and proteins of known function. This analysis allowed us to classify these 6 previously unannotated ORFans into their specific protein families: carboxylesterase/thioesterase, metal-dependent deacetylase, P-loop kinases, 3-methyladenine DNA glycosylase, BTB domain and eukaryotic translation initiation factor eIF4E.</p> <p>Conclusion</p> <p>Using stringent fold recognition criteria we have assigned three-dimensional structures for 21 of the ORFans encoded in the Mimivirus genome. Further, based on the 3D models and an analysis of the conservation of functionally important residues and motifs, we were able to derive functional attributes for 6 of the ORFans. Our computational identification of important functional sites in these ORFans can be the basis for a subsequent experimental verification of our predictions. Further computational and experimental studies are required to elucidate the 3D structures and functions of the remaining Mimivirus ORFans.</p

Crystal Structure of a Novel Esterase Rv0045c from Mycobacterium tuberculosis

There are at least 250 enzymes in Mycobacterium tuberculosis (M. tuberculosis) involved in lipid metabolism. Some of the enzymes are required for bacterial survival and full virulence. The esterase Rv0045c shares little amino acid sequence similarity with other members of the esterase/lipase family. Here, we report the 3D structure of Rv0045c. Our studies demonstrated that Rv0045c is a novel member of α/β hydrolase fold family. The structure of esterase Rv0045c contains two distinct domains: the α/β fold domain and the cap domain. The active site of esterase Rv0045c is highly conserved and comprised of two residues: Ser154 and His309. We proposed that Rv0045c probably employs two kinds of enzymatic mechanisms when hydrolyzing C-O ester bonds within substrates. The structure provides insight into the hydrolysis mechanism of the C-O ester bond, and will be helpful in understanding the ester/lipid metabolism in M. tuberculosis

Purification and Characterization of Organic Solvent and Detergent Tolerant Lipase from Thermotolerant Bacillus sp. RN2

The aim of this study was to characterize the organic solvent and detergent tolerant properties of recombinant lipase isolated from thermotolerant Bacillus sp. RN2 (Lip-SBRN2). The isolation of the lipase-coding gene was achieved by the use of inverse and direct PCR. The complete DNA sequencing of the gene revealed that the lip-SBRN2 gene contains 576 nucleotides which corresponded to 192 deduced amino acids. The purified enzyme was homogeneous with the estimated molecular mass of 19 kDa as determined by SDS-PAGE and gel filtration. The Lip-SBRN2 was stable in a pH range of 9–11 and temperature range of 45–60 °C. The enzyme was a non metallo-monomeric protein and was active against pNP-caprylate (C8) and pNP-laurate (C12) and coconut oil. The Lip-SBRN2 exhibited a high level of activity in the presence of 108% benzene, 102.4% diethylether and 112% SDS. It is anticipated that the organic solvent and detergent tolerant enzyme secreted by Bacillus sp. RN2 will be applicable as catalysts for reaction in the presence of organic solvents and detergents

SPODOBASE : an EST database for the lepidopteran crop pest Spodoptera

BACKGROUND: The Lepidoptera Spodoptera frugiperda is a pest which causes widespread economic damage on a variety of crop plants. It is also well known through its famous Sf9 cell line which is used for numerous heterologous protein productions. Species of the Spodoptera genus are used as model for pesticide resistance and to study virus host interactions. A genomic approach is now a critical step for further new developments in biology and pathology of these insects, and the results of ESTs sequencing efforts need to be structured into databases providing an integrated set of tools and informations. DESCRIPTION: The ESTs from five independent cDNA libraries, prepared from three different S. frugiperda tissues (hemocytes, midgut and fat body) and from the Sf9 cell line, are deposited in the database. These tissues were chosen because of their importance in biological processes such as immune response, development and plant/insect interaction. So far, the SPODOBASE contains 29,325 ESTs, which are cleaned and clustered into non-redundant sets (2294 clusters and 6103 singletons). The SPODOBASE is constructed in such a way that other ESTs from S. frugiperda or other species may be added. User can retrieve information using text searches, pre-formatted queries, query assistant or blast searches. Annotation is provided against NCBI, UNIPROT or Bombyx mori ESTs databases, and with GO-Slim vocabulary. CONCLUSION: The SPODOBASE database provides integrated access to expressed sequence tags (EST) from the lepidopteran insect Spodoptera frugiperda. It is a publicly available structured database with insect pest sequences which will allow identification of a number of genes and comprehensive cloning of gene families of interest for scientific community. SPODOBASE is available from URL

A study of carboxylic ester hydrolases: structural classification, properties, and database

The carboxylic ester hydrolases (CEHs) are enzymes that hydrolyze an ester bond to form a carboxylic acid and an alcohol. They are one of the enzyme groups that are most explored industrially for their applications in the food, flavor, pharmaceutical, organic synthesis, and detergent industries. We classified CEHs into families and clans according to their amino acid sequences (primary structures) and three-dimensional structures (tertiary structures). Our work has established the systematic structural classification of the CEHs. Primary structures of family members are similar to each other, and their active sites and reaction mechanisms are conserved. The tertiary structures of members of each clan, which is composed of different families, remain very similar, although amino acid sequences of members of different families are not similar. CEHs were divided into 127 families by use of BLAST, with 67 families being grouped into seven clans. Multiple sequence alignment and tertiary structures superposition were used, and active sites and reaction mechanisms were analyzed. Python and Shell scripts were implemented to automate the process of comparing CEH primary and tertiary structures. A comprehensive database, CASTLE (CArboxylic eSTer hydroLasEs), may be constructed to provide the primary and tertiary structures of CEHs. This database would be available at www.castle.enzyme.iastate.edu and will be accessible to the entire biology community

Large-scale identification of human genes implicated in epidermal barrier function

Identification of genes expressed in epidermal granular keratinocytes by ORESTES, including a number that are highly specific for these cells

Structure, Function and Evolution of Insect Carboxylesterases

Insect carboxylesterases (CBEs) have proven to be a highly adaptable family of enzymes that has undergone extensive functional diversification and sequence divergence over a short span of evolutionary time. This makes these enzymes ideal examples to explore the evolutionary processes that lead to the unique functions of enzymes. In this thesis I present two such examples. The first example is addressed in chapters 2 and 3: the evolution of insecticide resistance CBEs. These enzymes are implicated in the most common forms of insecticide resistance, a global issue that threatens both our agricultural productivity and health. While a great deal of work has gone into the identification of insecticide resistance CBEs, there has been little molecular characterization of these enzymes. This is vital to better understand how they function and to allow target-based inhibitor design to combat the resistance they provide. In chapter 2, I describe my attempts to express a large range of insecticide resistance CBEs in Eschericia coli. This is a critical first step in the large-scale expression required for crystallization and full characterization. I identified five insecticide resistance CBEs with sufficient expression for crystallization trials. In chapter 3, I describe the crystallization and characterization of one of these CBEs, CqestB2, which is the most common insecticide resistance CBE in the important disease vector, Culex quinquefasciatus. CqestB2 is the first insecticide sequestration CBE to be structurally characterized. Its structure demonstrates a high similarity to the insecticide target, acetylcholinesterase. Sequence similarity networks of all insect CBEs demonstrated that insecticide resistance CBEs share a level of similarity. This was further emphasized through a structural comparison between CqestB2 and other insect CBEs. Kinetic characterization of CqestB2 supported its role in organophosphate resistance via sequestration. Finally, a comparison between CqestB2 and its naturally occurring isoforms suggests target-based inhibitor design may have broad applicability. The second example is addressed in chapter 4: the evolution of an odorant degrading enzyme (ODE) from a juvenile hormone esterase (JHE) duplication in Drosophila melanogaster. While the evolution of new functions via gene duplication is a widely accepted mechanism, there are relatively few, well characterized examples of this process. The distinct regulation and substrate specificities of these enzymes also provides a unique opportunity to explore the interaction of both structural and regulatory changes in neofunctionalization. A phylogenetic analysis shows that JHEs have been the template to many distinct functional groups of enzymes. Biochemical comparison reveals sufficient promiscuity in the D. melanogaster JHE (DmJHE) to have immediate utility as an ODE. Homology modelling and comparison with known structures of insect JHEs and ODEs revealed similarities and differences that distinguish these groups and suggests key structural changes that explain this example of neofunctionalization. Finally, in chapter 5, I discuss the significance of my research and the insights that these two examples provide to the process of enzyme evolution. The first, the insecticide resistance CBEs provide a critical example of the early stages of enzyme evolution whereby a promiscuous activity results in a novel function. The comparisons drawn between CqestB2 and LcaE7, an insecticide resistance CBE from Lucilia cuprina that utilizes catalytic detoxification, emphasize distinct strategies through which natural evolution selects for novel functions. The second, DmJHE and DmJHE duplication, provides an example of a later stage in the process of neofunctionalization whereby structural and regulatory changes have resulted in two distinct enzymes with unique functions

Development of diagnostic tools for detecting expression of resistance-associated esterases in the tobacco budworm, Heliothis virescens (F.)

Esterase-based metabolic resistance was studied using biochemical and biological assays with organophosphate (OP)- and pyrethroid (PYR)- resistant tobacco budworms, Heliothis virescens. In biochemical assays, results suggest that: (1) esterase activities toward all substrates used were enhanced in both resistant strains compared with the susceptible strain, suggesting that esterases were involved in resistance; (2) esterase profiles differed depending on the strain and substrate used, and these differences were visualized by using native polyacrylamide gel electrophoresis; 3) esterase activities toward some pyrethroid substrates were significantly higher (P ≤ 0.01) in the PYR-R strain than those in the OP-R strain. These results suggest that pyrethroid substrates may be useful indicators for detecting esterases associated with pyrethroid resistance. Finally, biochemical assays were modified for use on solid materials, and esterase substrates were tested in filter paper assays. Whereas some differences in color intensity were detected between susceptible and resistant strains, these differences were not dramatic. Thus, utility of these substrates in such assays appears limited at this time, but further research is warranted. In biological assays, two approaches were taken to improve the precision with which esterases associated with pyrethroid resistance were detected. First, bioassays were used to test effects of pyrethroid substrates and traditional synergists (e.g., piperonyl butoxide) on insecticide toxicity. Non-toxic pyrethroid esters enhanced pyrethroid toxicity to a greater extent than DEF, a compound widely used as an esterase inhibitor. In addition, synergism of profenofos and cypermethrin toxicity in both resistant strains by an oxidase inhibitor, 1, 2, 4-trichloro-3 (2-propynyloxy) benzene, suggests that P450 monooxygenases were also involved in resistance. The second method tested was to utilize bioactivated insecticides to detect esterases. Absence of negative cross-resistance to insecticides (i.e., acephate and indoxacarb) that are activated by esterases suggests that detoxication of these compounds in resistant insects proceeds more rapidly than their activation by esterases. Finally, levels of cross-resistance to tefluthrin and trans-fenfluthrin were lower than to permethrin and cypermethrin in both resistant strains, suggesting that resistance to insecticides in which sites for detoxifying enzymes (e.g., oxidases) are blocked develops more slowly than resistance to those in which metabolic sites are present