Discovery and characterization of thermophilic limonene‐1,2‐epoxide hydrolases from hot spring metagenomic libraries

Abstract

The epoxide hydrolases (EHs) represent an attractive option for the synthesis of chiral epoxides and 1,2-diols which are valuable building blocks for the synthesis of several pharmaceutical compounds. A metagenomic approach has been used to identify two new members of the atypical EH limonene-1,2-epoxide hydrolase (LEH) family of enzymes. These two LEHs (Tomsk-LEH and CH55-LEH) show EH activities towards different epoxide substrates, differing in most cases from those previously identified for Rhodococcus erythropolis (Re-LEH) in terms of stereoselectivity. Tomsk-LEH and CH55-LEH, both from thermophilic sources, have higher optimal temperatures and apparent melting temperatures than Re-LEH. The new LEH enzymes have been crystallized and their structures solved to high resolution in the native form and in complex with the inhibitor valpromide for Tomsk-LEH and poly(ethylene glycol) for CH55-LEH. The structural analysis has provided insights into the LEH mechanism, substrate specificity and stereoselectivity of these new LEH enzymes, which has been supported by mutagenesis studies. DatabaseThe atomic coordinates and structure factors of the crystal structures have been deposited in the Protein Data Bank with the codes 5AIF (Tomsk-LEH native structure), 5AIG (Tomsk-LEH valpromide complex), 5AIH (CH55-LEH native structure) and 5AII (CH55-LEH PEG complex). Nucleotide sequence data are available in the GenBank databases under the accession numbers KP765711 (Tomsk-LEH) and KP765710 (CH55-LEH).Abbreviations CH55-LEH, limonene-1,2-epoxide hydrolase from the metagenomic DNA from the Chinese sample; de, diastereomeric excess; ee, enantiomeric excess; EH, epoxide hydrolase; LEH, limonene-1,2-epoxide hydrolase; Mt-LEH, Mycobacterium tuberculosis limonene-1,2-epoxide hydrolase; PEG, poly(ethylene glycol); Re-LEH, Rhodococcus erythropolis limonene-1,2-epoxide hydrolase; Tomsk-LEH, limonene-1,2-epoxide hydrolase from the metagenomic DNA from the Tomsk sample.

Efficient Epoxide Hydrolase Catalyzed Resolutions of (+)‐ and (−)‐cis/trans‐Limonene Oxides

“…Recently, we have discovered and characterized two novel LEHs (Tomsk‐LEH and CH55‐LEH) from hot‐springs metagenomic libraries . In agreement with their natural sources, both enzymes, which have been cloned and overexpressed in Escherichia coli , have higher optimal temperatures and apparent melting temperatures than Re ‐LEH.…”

Metagenomic Analysis of Hot Springs in Central India Reveals Hydrocarbon Degrading Thermophiles and Pathways Essential for Survival in Extreme Environments

“…(Mardanov et al, 2011; Menzel et al, 2015). Many studies have also revealed the presence of genes encoding for enzymes of biotechnological interest, such as hydrolases, xylanases, proteases, galactosidases, and lipases (Ferrandi et al, 2015; Littlechild, 2015). Some studies focusing on the metagenomic analysis of hot springs located in India have reported Bacillus licheniformis (Mangrola et al, 2015), Bacillus megaterium, Bacillus sporothermodurans, Hydrogenobacter sp., Thermus thermophilus, Thermus brockianus (Bhatia et al, 2015), Clostridium bifermentans, Clostridium lituseburense (Ghelani et al, 2015), Opitutus terrae, Rhodococcus erythropolis , and Cellovibrio mixtus (Mehetre et al, 2016) as major bacterial genera.…”

Improving the ‘tool box’ for robust industrial enzymes

“…A bioinformatic approach was used to identify the genes coding for these industrially important enzymes using the mesophilic Rhodococcus LEH as a search. The two new thermophilic LEHs have been cloned and over-expressed in E. coli and the resultant proteins characterised both biochemically and structurally [19]. The atomic coordinates and structure factors of the crystal structures obtained have been deposited in the PDB: 5AIF (Tomsk-LEH native structure), 5AIG (Tomsk-LEH valpromide complex), 5AIH (CH55-LEH native structure) and 5AII (CH55-LEH PEG complex).…”