Unraveling Energy and Dynamics Determinants to Interpret Protein Functional Plasticity: The Limonene-1,2-epoxide-hydrolase Case Study.

The balance between structural stability and functional plasticity in proteins that share common three-dimensional folds is the key factor that drives protein evolvability. The ability to distinguish the parts of homologous proteins that underlie common structural organization patterns from the parts acting as regulatory modules that can sustain modifications in response to evolutionary pressure may provide fundamental insights for understanding sequence-structure-dynamics relationships. In applicative terms, this would help develop rational protein design methods. Herein, we apply recently developed computational methods, validated by experimental tests, to address these questions in a set of homologous enzymes representative of the limonene-1,2-epoxide-hydrolase family (LEH) characterized by different stabilities, namely Rhodococcus erythropolis LEH (Re-LEH), Tomks-LEH, CH55-LEH, and the two thermostable Re-LEH variants Re-LEH-F1b and Re-LEH-P. Our results show that these enzymes, despite significant sequence variations, exploit a few highly conserved stabilization determinants to guarantee structural stability linked to biological functionality. Multiple sequence analysis shows that these key elements are also shared by a larger set of LEHs structural homologues, despite very low sequence identity and functional diversity. In this framework, stabilizing elements that we hypothesize to have an accessory role are characterized by a lower degree of sequence identity and higher mutability. We suggest that our approach can be successfully used to pinpoint the distinctive energy fingerprint of a class of proteins as well as to locate those modulators whose modification could be exploited to tune protein stability and dynamic properties.

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

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.