The structure of a novel thermophilic esterase from the planctomycetes species, thermogutta terrifontis reveals an open active site due to a minimal 'cap' domain.

Published under the CC-BY license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and the source are credited.A carboxyl esterase (TtEst2) has been identified in a novel thermophilic bacterium, Thermogutta terrifontis from the phylum Planctomycetes and has been cloned and over-expressed in Escherichia coli. The enzyme has been characterized biochemically and shown to have activity toward small p-nitrophenyl (pNP) carboxylic esters with optimal activity for pNP-acetate. The enzyme shows moderate thermostability retaining 75% activity after incubation for 30 min at 70°C. The crystal structures have been determined for the native TtEst2 and its complexes with the carboxylic acid products propionate, butyrate, and valerate. TtEst2 differs from most enzymes of the α/β-hydrolase family 3 as it lacks the majority of the 'cap' domain and its active site cavity is exposed to the solvent. The bound ligands have allowed the identification of the carboxyl pocket in the enzyme active site. Comparison of TtEst2 with structurally related enzymes has given insight into how differences in their substrate preference can be rationalized based upon the properties of their active site pockets.Hotzyme ProjectEuropean Union 7th Framework ProgrammeBiotechnology and Biological Sciences Research Council (BBSRC

Structural, Kinetic and Mutational Analysis of Two Bacterial Carboxylesterases

The crystal structures of two thermostable carboxylesterase Est30 and Est55 from Geobacillus stearothermophilus were determined to help understand their functions and applications in industry or medicine. The crystal structure of Est30 was determined at 1.63 Å resolution by the multiple anomalous dispersion method. The two-domain Est30 structure showed a large domain with a modified alpha/beta hydrolase core including a seven, rather than an eight-stranded beta sheet, and a smaller cap domain comprising three alpha helices. A 100 Da tetrahedral ligand, propyl acetate, was observed to be covalently bound to the side chain of Ser94 in the catalytic triad. This ligand complex represents the first tetrahedral intermediate in the reaction mechanism. Therefore, this Est30 crystal structure will help understand the mode of action of all enzymes in the serine hydrolase superfamily. Est55 is a bacterial homologue of the mammalian carboxylesterases involved in hydrolysis and detoxification of numerous peptides and drugs and in prodrug activation. Est55 crystals were grown at pH 6.2 and pH 6.8 and the structures were determined at resolutions of 2.0 and 1.58 Å respectively. Est55 folds into three domains, a catalytic domain, an α/β domain and a regulatory domain. This structure is in an inactive form; the side chain of His409, one of the catalytic triad residues, is pointing away from the active site. Moreover, the adjacent Cys408 is triply oxidized and lies in the oxyanion hole, which would block the entry of substrate to its binding site. This structure suggested a self-inactivation mechanism, however, Cys408 is not essential for enzyme activity. Mutation of Cys408 showed that hydrophobic side chains at this position were favorable, while polar serine was unfavorable for enzyme activity. Both Est30 and Est55 were shown to hydrolyze the prodrug CPT-11 into the active form SN-38. Therefore, Est30 and Est55 are potential candidates for use with irinotecan in cancer therapy. The catalytic efficiency (kcat/Km) of Est30 is about 10-fold lower than that of Est55. The effects of the Cys408 substitutions on Est55 activity differed for the two substrates, p-NP butyrate and CPT-11. Mutant C408V may provide a more stable form of Est55

Molecular analysis of olfactory perception in Drosophila melanogaster

Over the last decade the insect olfactory system has emerged as an important model system with which to investigate the biochemical basis of eukaryote signalling processes. It is believed that certain odorant degrading enzymes are required to maintain the ongoing sensitivity of an insect’s olfactory neuronal system by repriming neurons. However, relatively few ODEs have been identified and characterized to date, especially in the model insect Drosophila melanogaster. The study presented here takes biochemical, neurobiological and behavioural approaches to elucidate the role of ODEs in D. melanogaster. After a review of relevant literature in Chapter 1, Chapter 2 decodes the antennal transcriptome of D. melanogaster for the first time. Using high quality genome sequence and transcriptomic data for many other tissues of this species already available, I identified a few antennae-selective esterases, cytochrome P450s (P450s), glutathione S-transferases (GSTs) and UDP-glycosyltransferases (UGTs). Of these, the activity of one esterase JHEdup, against a range of volatile odorants was found to be comparable with other known ODEs from different species, mainly Lepidoptera. I also identified the presence of another esterase, EST6, at high levels in the antennae. It has previously been proposed that EST6 is a catalyst for the transformation of pheromonal and kairomonal esters to the corresponding alcohols and acids, thereby mediating various mating behaviours. I further examined the proposed effect of EST6 by comparing wild type and EST6 null flies at a neurobiological and behavioural level. The findings, presented in Chapter 3, show this enzyme is important for the flies to respond to incoming volatile odorants and affects their subsequent behaviours. Additionally, EST6 has previously been reported to hydrolyse cis-vaccenyl acetate (cVA), the major pheromone known in Drosophila, in vitro and recent electroantennogram (EAG) experiments with EST6 wild type and null flies exposed to cVA suggest that this might also be an in vivo function. I therefore conducted experiments to understand the biochemical activity of EST6 against cVA. I also measured its activity against 84 other bioactive esters. The results categorically show that EST6 has no activity against cVA but has very good activity against a wide range of fruit- and yeast-derived volatiles known to play a role in mediating female reproductive behaviour. These results are presented in Chapter 4, along with a crystal structure of EST6. The final chapter of this thesis then discusses the overall findings of these studies and offers a broader perspective on future directions. The three major conclusions from the work are as follows. Firstly, JHEdup and EST6 are broad range ODEs active against a wide range of food odorants. Secondly, EST6 may also have a role in cVA processing but not actually as an ODE against this substrate. Thirdly, ODEs may be a fruitful system to develop biocontrol systems for pest insects based on disrupting their olfactory system

Multiple forms of carboxylesterase from Leptinotarsa decemlineata hemolymph associated with permethrin resistance.

The purpose of this dissertation is to purify and characterize the carboxylesterase(s) associated with permethrin resistance in the permethrin-resistant (PE-R) strain of Colorado potato beetle (CPB), Leptinotarsa decemlineata, and to develop an immunoassay system for the detection of resistance in field populations of CPB. Most carboxylesterase (CbE) activity is found in the hemolymph and the soluble fraction of body tissue. Among a number of charged forms of CbE identified from hemolymph, the pI 4.5-4.9 CbEs are quantitatively elevated and are most responsible for permethrin resistance in the PE-R strain. Permethrin CbEs (i.e., pI 4.2-4.8 CbEs) have been purified from the hemolymph of the PE-R strain through several chromatographic procedures. The pI 4.8 CbE is a 46-48 kDa monomeric protein. The pi 4.5 CbE is likely a 57-59 kDa dimeric protein. All pI 4.5-4.8 forms are glycoproteins but the charge heterogeneity is not associated with N-glycan moieties. Biochemical properties of the pI 4.2-4.5 CbEs have been comparatively characterized through substrate kinetic analyses, specific inhibition studies, and pH-temperature experiments. The pI 4.8 and 4.5 CbEs share a number of similarities in their biochemical properties and functional role in resistance despite of their distinct molecular properties. The kinetics of inhibition of the pI 4.5-4.8 CbEs by permethrin and DDT are best described by a mixed-noncompetitive type and a noncompetitive type inhibition, respectively. The kinetic analyses indicate the presence of hydrophobic non-catalytic site(s) as well as hydrophobic catalytic site(s) that are available for the binding to hydrophobic insecticides. Along with a low level of permethrin hydrolysis, the hydrophobic binding nature of the pI 4.5-4.8 CbEs suggests that permethrin resistance is mainly conferred by sequestration rather than rapid hydrolysis of permethrin. The nonspecific sequestration by the pI 4.5-4.8 CbEs appears to be associated with the cross-resistance of the PE-R strain to other hydrophobic insecticides such as other pyrethroids, DDT, and abamectin. Polyclonal antisera have been generated against the 30, 48, and 60 kDa denatured CbE immunogens. A high degree of cross-reactivities of the antisera to different immunogens indicate that all CbE immunogens share a high level of structural similarity. An antibody capture immunoassay using denatured CPB hemolymph is shown to be effective in detecting the different levels of permethrin CbE in permethrin-resistant and -susceptible populations of CPB

Enzyme Dynamic in Plant Nutrition Uptake and Plant Nutrition

Soil contains enzymes, constantly interacting with soil constituents, e.g. minerals, rhizosphere and numerous nutrients. Enzymes, in turn, catalyse important biochemical reactions for rhizobacteria and plants, stabilize the soil by degrading wastes and mediate nutrient recycling.The available enzymes inside soil could originate from plants, animals or microbes. The enzymes that are produced from these organism could exhibit intracellular activities, at the cell membrane, interacting therefore with soil and its constituents, or extracellularly (so freely available). Therefore, vis-à-vis to plant nutrition, the (extra or sub) cellular localization has a key role. Typical major enzymes available in soil can be listed as dehydrogenases, hydrogenases, oxidases, catalases, peroxidases, phenol o-hydroxylase, dextransucrase, aminotransferase, rhodanese, carboxylesterase, lipase, phosphatase, nuclease, phytase, arylsulphatase, amylase, cellulase, inulase, xylanase, dextranase, levanase, poly-galacturonase, glucosidase, galactosidase, invertase, peptidase, asparaginase, glutaminase, amidase, urease, aspartate decarboxylase, glutamate decarboxylase and aromatic amino acid decarboxylase. An interesting strategy for improving the nutritional quality of the soil would be to inoculate microorganism to soil while giving attention to mineral or other compounds that affect enzyme activity in soil. Since, some elements or compounds could show both activation and inhibitory effect, such as Fe, Na, etc. metals, the regulation of their bioavailability is crucial

Analysis of the Genome of the Sexually Transmitted Insect Virus Helicoverpa zea Nudivirus 2

The sexually transmitted insect virus Helicoverpa zea nudivirus 2 (HzNV-2) was determined to have a circular double-stranded DNA genome of 231,621 bp coding for an estimated 113 open reading frames (ORFs). HzNV-2 is most closely related to the nudiviruses, a sister group of the insect baculoviruses. Several putative ORFs that share homology with the baculovirus core genes were identified in the viral genome. However, HzNV-2 lacks several key genetic features of baculoviruses including the late transcriptional regulation factor, LEF-1 and the palindromic hrs, which serve as origins of replication. The HzNV-2 genome was found to code for three ORFs that had significant sequence homology to cellular genes which are not generally found in viral genomes. These included a presumed juvenile hormone esterase gene, a gene coding for a putative zinc-dependent matrix metalloprotease, and a major facilitator superfamily protein gene; all of which are believed to play a role in the cellular proliferation and the tissue hypertrophy observed in the malformation of reproductive organs observed in HzNV-2 infected corn earworm moths, Helicoverpa zea

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

Screening and Characterization of Novel Polyesterases from Environmental Metagenomes with High Hydrolytic Activity against Synthetic Polyesters

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