Dataset Information

Recombinant S. cerevisiae expressing Old Yellow Enzymes from non-conventional yeasts: an easy system for selective reduction of activated alkenes.

ABSTRACT: BACKGROUND: Old Yellow Enzymes (OYEs) are flavin-dependent enoate reductases (EC 1.6.99.1) that catalyze the stereoselective hydrogenation of electron-poor alkenes. Their ability to generate up to two stereocenters by the trans-hydrogenation of the C?=?C double bond is highly demanded in asymmetric synthesis. Isolated redox enzymes utilization require the addition of cofactors and systems for their regeneration. Microbial whole-cells may represent a valid alternative combining desired enzymatic activity and efficient cofactor regeneration. Considerable efforts were addressed at developing novel whole-cell OYE biocatalysts, based on recombinant Saccharomyces cerevisiae expressing OYE genes. RESULTS: Recombinant S. cerevisiae BY4741?Oye2 strains, lacking endogenous OYE and expressing nine separate OYE genes from non-conventional yeasts, were used as whole-cell biocatalysts to reduce substrates with an electron-poor double bond activated by different electron-withdrawing groups. Ketoisophorone, ?-methyl-trans-cinnamaldehyde, and trans-?-methyl-?-nitrostyrene were successfully reduced with high rates and selectivity. A series of four alkyl-substituted cyclohex-2-enones was tested to check the versatility and efficiency of the biocatalysts. Reduction of double bond occurred with high rates and enantioselectivity, except for 3,5,5-trimethyl-2-cyclohexenone. DFT (density functional theory) computational studies were performed to investigate whether the steric hindrance and/or the electronic properties of the substrates were crucial for reactivity. The three-dimensional structure of enoate reductases from Kluyveromyces lodderae and Candida castellii, predicted through comparative modeling, resulted similar to that of S. cerevisiae OYE2 and revealed the key role of Trp116 both in substrate specificity and stereocontrol. All the modeling studies indicate that steric hindrance was a major determinant in the enzyme reactivity. CONCLUSIONS: The OYE biocatalysts, based on recombinant S. cerevisiae expressing OYE genes from non-conventional yeasts, were able to differently reduce the activated double bond of enones, enals and nitro-olefins, exhibiting a wide range of substrate specificity. Moreover whole-cells biocatalysts bypassed the necessity of the cofactor recycling and, tuning reaction parameters, allowed the synthetic exploitation of endogenous carbonyl reductases. Molecular modeling studies highlighted key structural features for further improvement of catalytic properties of OYE enzymes.

SUBMITTER: Romano D

PROVIDER: S-EPMC4013436 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Recombinant S. cerevisiae expressing Old Yellow Enzymes from non-conventional yeasts: an easy system for selective reduction of activated alkenes.

Romano Diego D Contente Martina Letizia ML Molinari Francesco F Eberini Ivano I Ruvutuso Erica E Sensi Cristina C Amaretti Alberto A Rossi Maddalena M Raimondi Stefano S

Microbial cell factories 20140425

<h4>Background</h4>Old Yellow Enzymes (OYEs) are flavin-dependent enoate reductases (EC 1.6.99.1) that catalyze the stereoselective hydrogenation of electron-poor alkenes. Their ability to generate up to two stereocenters by the trans-hydrogenation of the C = C double bond is highly demanded in asymmetric synthesis. Isolated redox enzymes utilization require the addition of cofactors and systems for their regeneration. Microbial whole-cells may represent a valid alternative combining desired enz ...[more]

PMID: 24767246

Similar Datasets

Project description:The reduction of C=X (X = N, O) bonds is a cornerstone in both synthetic organic chemistry and biocatalysis. Conventional reduction mechanisms usually involve a hydride ion targeting the less electronegative carbon atom. In a departure from this paradigm, our investigation into Old Yellow Enzymes (OYEs) reveals a mechanism involving transfer of hydride to the formally more electronegative nitrogen atom within a C=N bond. Beyond their known ability to reduce electronically activated C=C double bonds, e.g., in α, β-unsaturated ketones, these enzymes have recently been shown to reduce α-oximo-β-ketoesters to the corresponding amines. It has been proposed that this transformation involves two successive reduction steps and proceeds via imine intermediates formed by the reductive dehydration of the oxime moieties. We employ advanced quantum mechanics/molecular mechanics (QM/MM) simulations, enriched by a two-tiered approach incorporating QM/MM (UB3LYP-6-31G*/OPLS2005) geometry optimization, QM/MM (B3LYP-6-31G*/amberff19sb) steered molecular dynamics simulations, and detailed natural-bond-orbital analyses to decipher the unconventional hydride transfer to nitrogen in both reduction steps and to delineate the role of active site residues as well as of substituents present in the substrates. Our computational results confirm the proposed mechanism and agree well with experimental mutagenesis and enzyme kinetics data. According to our model, the catalysis of OYE involves hydride transfer from the flavin cofactor to the nitrogen atom in oximoketoesters as well as iminoketoesters followed by protonation at the adjacent oxygen or carbon atoms by conserved tyrosine residues and active site water molecules. Two histidine residues play a key role in the polarization and activation of the C=N bond, and conformational changes of the substrate observed along the reaction coordinate underline the crucial importance of dynamic electron delocalization for efficient catalysis.

Dataset Information

Recombinant S. cerevisiae expressing Old Yellow Enzymes from non-conventional yeasts: an easy system for selective reduction of activated alkenes.

Publications

Recombinant S. cerevisiae expressing Old Yellow Enzymes from non-conventional yeasts: an easy system for selective reduction of activated alkenes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets