Two new ene-reductases from photosynthetic extremophiles enlarge the panel of old yellow enzymes: CtOYE and GsOYE

Abstract

Looking for new ene-reductases with uncovered features beneficial for biotechnological applications, by mining genomes of photosynthetic extremophile organisms, we identified two new Old Yellow Enzyme homologues: CtOYE, deriving from the cyanobacterium Chroococcidiopsis thermalis, and GsOYE, from the alga Galdieria sulphuraria. Both enzymes were produced and purified with very good yields and displayed catalytic activity on a broad substrate spectrum by reducing α,β-unsaturated ketones, aldehydes, maleimides and nitroalkenes with good to excellent stereoselectivity. Both enzymes prefer NADPH but demonstrate a good acceptance of NADH as cofactor. CtOYE and GsOYE represent robust biocatalysts showing high thermostability, a wide range of pH optimum and good co-solvent tolerance. High resolution X-ray crystal structures of both enzymes have been determined, revealing conserved features of the classical OYE subfamily as well as unique properties, such as a very long loop entering the active site or an additional C-terminal alpha helix in GsOYE. Not surprisingly, the active site of CtOYE and GsOYE structures revealed high affinity toward anions caught from the mother liquor and trapped in the anion hole where electron-withdrawing groups such as carbonyl group are engaged. Ligands (para-hydroxybenzaldehyde and 2-methylcyclopenten-1-one) added on purpose to study complexes of GsOYE were detected in the enzyme catalytic cavity, stacking on top of the FMN cofactor, and support the key role of conserved residues and FMN cofactor in the catalysis.

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

“…The protein was eluted in 50 mM Tris-HCl pH 8.0, 250 mM imidazole solution. Enzyme purity was checked by SDS-PAGE and immunoblotting, while the concentration was evaluated by spectrophotometric measurement of free flavin cofactor titer in a solution of thermal denatured protein, calculated as previously reported [ 21 ].…”

“…Apparent unfolding temperatures of the recombinant enzyme, T m , in standard conditions (50 mM Tris-HCl pH 8.0) and in the presence of different co-solvents (ethanol, acetonitrile and dimethyl sulfoxide) at different percentages (5–40% v /v ), were determined using the Thermofluor method as described before [ 21 ]. Ca OYE purified protein was used diluted to 5 μM in 50 mM Tris-HCl buffer pH 8.0.…”

“…CA49 [ 19 ], FOYE-1 from the acidophilic iron-oxidizing bacterium Ferrovum sp. JA12 [ 20 ], Ct OYE from the thermophilic cyanobacterium Chroococcidiopsis thermalis and Gs OYE from the polyextremophilic alga Galdieria sulphuraria [ 21 ].…”

“…Aiming at increasing the protein diversity of this class of enzymes, by using as query the sequence of YqjM (from Bacillus subtilis ) that is conventionally considered a representative member for the thermophilic-like subclasses of OYEs (Class II or Class III, following Peters et al [ 22 ] or Böhmer et al [ 23 ], respectively), we initiated a search for OYE homologues in unconventional organisms such as photosynthetic extremophile bacteria. To our knowledge, indeed, only higher plants [ 24 , 25 , 26 ], few examples of cyanobacteria [ 21 , 27 , 28 ] and two algae [ 21 , 23 ] have been explored so far as sources of ene-reductases. Among the putative enzymes identified, we selected one protein from Chloroflexus aggregans .…”

Characterization of the Novel Ene Reductase Ppo-Er1 from Paenibacillus Polymyxa

“…Overall, Ppo-Er1 s specific activity for other typical ene reductase substrates such as carvone (k cat /K m = 0.5 mM −1 s −1 ) and cyclohexanone (k cat /K m = 0.4 mM −1 s −1 ) was found to be in a similar range as those described for other well-known OYEs such as the classical PETNR (carvone: k cat /K m = 2 mM −1 s −1 ; cyclohexanone: k cat /K m = 5 mM −1 s −1 ) [50] and the thermophilic-like YqjM (cyclohexanone: k cat /K m = 6.4 mM −1 s −1 ) [46] (Table 2). Maleimide, however, is better converted by ene reductases from photosynthetic extremophiles such as CtOYE (k cat /K m = 1940 mM −1 s −1 ) or GsOYE (k cat /K m = 399 mM −1 s −1 ) [52] the thermophilic-like OYERo2 (k cat /K m = 10,800 mM −1 s −1 ) [53] or the class III OYE YqiG (k cat /K m = 800 mM −1 s −1 ) ( Table 2). Table 2.…”