An epoxide hydrolase from endophytic Streptomyces shows unique structural features and wide biocatalytic activity.

The genus Streptomyces is characterized by the production of a wide variety of secondary metabolites with remarkable biological activities and broad antibiotic capabilities. The presence of an unprecedented number of genes encoding hydrolytic enzymes with industrial appeal such as epoxide hydrolases (EHs) reveals its resourceful microscopic machinery. The whole-genome sequence of Streptomyces sp. CBMAI 2042, an endophytic actinobacterium isolated from Citrus sinensis branches, was explored by genome mining, and a putative α/ß-epoxide hydrolase named B1EPH2 and encoded by 344 amino acids was selected for functional and structural studies. The crystal structure of B1EPH2 was obtained at a resolution of 2.2 Šand it was found to have a similar fold to other EHs, despite its hexameric quaternary structure, which contrasts with previously solved dimeric and monomeric EH structures. While B1EPH2 has a high sequence similarity to EHB from Mycobacterium tuberculosis, its cavity is similar to that of human EH. A group of 12 aromatic and aliphatic racemic epoxides were assayed to determine the activity of B1EPH2; remarkably, this enzyme was able to hydrolyse all the epoxides to the respective 1,2-diols, indicating a wide-range substrate scope acceptance. Moreover, the (R)- and (S)-enantiomers of styrene oxide, epichlorohydrin and 1,2-epoxybutane were used to monitor enantiopreference. Taken together, the functional and structural analyses indicate that this enzyme is an attractive biocatalyst for future biotechnological applications.

Crystal structures of the closed form of Mycobacterium tuberculosis dihydrofolate reductase in complex with dihydrofolate and antifolates.

Tuberculosis is a disease caused by Mycobacterium tuberculosis and is the leading cause of death from a single infectious pathogen, with a high prevalence in developing countries in Africa and Asia. There still is a need for the development or repurposing of novel therapies to combat this disease owing to the long-term nature of current therapies and because of the number of reported resistant strains. Here, structures of dihydrofolate reductase from M. tuberculosis (MtDHFR), which is a key target of the folate pathway, are reported in complex with four antifolates, pyrimethamine, cycloguanil, diaverdine and pemetrexed, and its substrate dihydrofolate in order to understand their binding modes. The structures of all of these complexes were obtained in the closed-conformation state of the enzyme and a fine structural analysis indicated motion in key regions of the substrate-binding site and different binding modes of the ligands. In addition, the affinities, through Kd measurement, of diaverdine and methotrexate have been determined; MtDHFR has a lower affinity (highest Kd) for diaverdine than pyrimethamine and trimethoprim, and a very high affinity for methotrexate, as expected. The structural comparisons and analysis described in this work provide new information about the plasticity of MtDHFR and the binding effects of different antifolates.

Data set of optimal parameters for colorimetric red assay of epoxide hydrolase activity.

The data presented in this article are related to the research article entitled "Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization" [1]. Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to the corresponding vicinal diols. This article describes the optimal parameters for the colorimetric red assay to determine the enzymatic activity, with an emphasis on the characterization of the kinetic parameters, pH optimum and thermal stability of this enzyme. The effects of reagents that are not resistant to oxidation by sodium periodate on the reactions can generate false positives and interfere with the final results of the red assay.

Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization.

Epoxide hydrolases (EHs) are enzymes that are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EHs have biotechnological potential in chiral chemistry. We report the cloning, purification, enzymatic activity, and conformational analysis of the TrEH gene from Trichoderma reesei strain QM9414 using circular dichroism spectroscopy. The EH gene has an open reading frame encoding a protein of 343 amino acid residues, resulting in a molecular mass of 38.2kDa. The enzyme presents an optimum pH of 7.2, and it is highly active at temperatures ranging from 23 to 50°C and thermally inactivated at 70°C (t1/2=7.4min). The Michaelis constants (Km) were 4.6mM for racemic substrate, 21.7mM for (R)-(+)-styrene oxide and 3.0mM for (S)-(-)-styrene oxide. The kcat/Km analysis indicated that TrEH is enantioselective and preferentially hydrolyzes (S)-(-)-styrene oxide. The conformational stability studies suggested that, despite the extreme conditions (high temperatures and extremely acid and basic pHs), TrEH is able to maintain a considerable part of its regular structures, including the preservation of the native cores in some cases. The recombinant protein showed enantioselectivity that was distinct from other fungus EHs, making this protein a potential biotechnological tool.