A New Thermophilic Ene-Reductase from the Filamentous Anoxygenic Phototrophic Bacterium Chloroflexus aggregans.

Marina Simona Robescu,Elisabetta Bergantino,Giovanni Loprete,Laura Cendron,Mattia Niero

doi:10.3390/microorganisms9050953

Marina Simona Robescu, Elisabetta Bergantino + Show 3 more

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https://doi.org/10.3390/microorganisms9050953

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Journal: Microorganisms	Publication Date: Apr 28, 2021
Citations: 7	License type: CC BY 4.0

Affiliation: University of Padua

Abstract

Aiming at expanding the biocatalytic toolbox of ene-reductase enzymes, we decided to explore photosynthetic extremophile microorganisms as unique reservoir of (new) biocatalytic activities. We selected a new thermophilic ene-reductase homologue in Chloroflexus aggregans, a peculiar filamentous bacterium. We report here on the functional and structural characterization of this new enzyme, which we called CaOYE. Produced in high yields in recombinant form, it proved to be a robust biocatalyst showing high thermostability, good solvent tolerance and a wide range of pH optimum. In a preliminary screening, CaOYE displayed a restricted substrate spectrum (with generally lower activities compared to other ene-reductases); however, given the amazing metabolic ductility and versatility of Chloroflexus aggregans, further investigations could pinpoint peculiar chemical activities. X-ray crystal structure has been determined, revealing conserved features of Class III (or thermophilic-like group) of the family of Old Yellow Enzymes: in the crystal packing, the enzyme was found to assemble as dimer even if it behaves as a monomer in solution. The description of CaOYE catalytic properties and crystal structure provides new details useful for enlarging knowledge, development and application of this class of enzymes.

Highlights

Microorganisms that live in extreme environments produce extremozymes, enzymes that have developed molecular mechanisms of adaptation to extreme physico-chemical conditions
A hypothetical protein from Chloroflexus aggregans (WP_015941499.1), annotated with an Old Yellow Enzyme (OYE)-like FMN binding domain, was identified by a tBLASTn search restricted to photosynthetic extremophiles, using the sequence of YqjM from B. subtilis (P54550) as query for thermophilic-like OYE subclass or Class III
A Clustal Omega alignment with all other Class III enzymes pointed out the catalytic residues and the finger print motifs of thermophilic-like OYEs, as reported by [37] (Figure S1)

Summary

Introduction

Microorganisms that live in extreme environments produce extremozymes, enzymes that have developed molecular mechanisms of adaptation to extreme physico-chemical conditions. Such properties make them very attractive as biocatalysts in industrial biotransformations [1], since industrial processes often require robust enzymes able to operate under challenging conditions such as high substrate concentration and temperature, farthest values of pH or salinity. Are nicotinamide-dependent flavoproteins catalyzing the asymmetric hydrogenation of a wide panel of activated alkenes (e.g., α,β-unsaturated ketones, aldehydes, nitroalkenes, carboxylic acids, and derivatives) (Scheme 1) They are ubiquitous in nature and have been found in yeasts, bacteria, plants and parasitic eukaryotes [2].

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