STRUCTURAL AND FUNCTIONAL CONSEQUENCES OF CIRCULAR PERMUTATION ON THE ACTIVE SITE OF OLD YELLOW ENZYME

Abstract

Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) can significantly enhance the enzyme's catalytic performance. Termini relocation into four regions of the protein (sectors I-IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I-III for further characterization by biophysical methods and X-ray crystallography. These investigations not only show trends in enzyme stability and quaternary structure as a function of termini location, but also provide a possible explanation for the catalytic gains in our top-performing OYE variant (new N-terminus at residue 303; sector III). Crystallographic analysis indicates that termini relocation into sector III affects the loop β6 region (amino acid positions: 290-310) of OYE1 which forms a lid over the active site. Peptide backbone cleavage greatly enhances local flexibility, effectively converting the loop into a tether and consequently increasing the environmental exposure of the active site. Interestingly, such active site remodeling does not negatively impact the enzyme's activity and stereoselectivity, nor does it perturb the conformation of other key active site residues with the exception of Y375. These observations were confirmed in truncation experiments, deleting all residues of the loop β6 region in our OYE variant. Intrigued by the finding that circular permutation leaves most of the key catalytic residues unchanged, we also tested OYE permutants for possible additive or synergistic effects of amino acid substitutions. Distinct functional changes in these OYE variants were detected upon mutations at W116, known in native OYE1 to cause inversion of diastereo-selectivity for (S)-carvone reduction. Our findings demonstrate the contribution of loop β6 toward determining the stereoselectivity of OYE1, an important insight for future OYE engineering efforts.

Keywords: Old Yellow Enzyme; X-ray crystallography; biocatalysis; circular permutation; oxidoreductases; protein engineering.

Scheme 1. Flavin-Dependent ene Reduction

Figure 1

Structure overlay of OYE1 (white; PDB: 1OYA), cpOYE154 (gray; PDB: 4RNX), and cpOYE303 (blue; PDB: 4RNU). Super positioning of the three structures was based on the FMN cofactor, shown in sticks. The three helices at the dimer interface (α4, α5, and α6) and the native OYE1 termini are labeled. The two inserts focus on the new termini regions in cpOYE154 (right) and cpOYE303 (left). Red arrows mark the locations of backbone cleavage (shown on OYE1) upon circular permutation, illustrating the portions of protein sequence invisible in the CP variants due to a lack of electron density.

Figure 2

Structural study of the active site of cpOYE303 (PBD: 4RNV) with key amino acid side chains (orange), FMN (white), and substrate analog p-hydroxybenzaldehyde (HBA; gray). (A) In two of the four protein complexes per asymmetric unit, the best fit to the observed electron density orients the aldehyde group of HBA in hydrogen-bonding distance to H191 and N194, whereas in the other two complexes (B), the ligand is flipped with its hydroxyl moiety pointing toward H191 and N194, as seen for OYE1. The gray mesh represents the 2mFo-DFc map contoured at the 1.0 σ level for key protein side chains, FMN and HBA. Hydrogen bonding distances (in angstroms) are indicated.

Figure 3

Comparison of active site binding pockets for (A) OYE1 (PDB: 1OYA), (B) cpOYE303 (PDB: 4RNU), and (C) cpOYE154 (PDB: 4RNX). Gray-shaded surfaces mark the interior protein surface. Key amino acids in the active sites (W116, H191, N194, and Y196) are orange. The bound FMN cofactor and substrate analog HBA (present in A and B) are shown as white sticks.

Figure 4

Conformational changes at positions 116, 296, and 375 upon substrate binding in native OYE1 and OYE variants. Ligands are highlighted in orange. (A) Overlay of OYE1 (blue, PDB: 1OYA) and OYE1 with bound HBA (gray, PDB: 1K03) shows the reorientation of F296 (90° rotation of phenyl ring) and Y375 (side chain rotation). (B) OYE1(W116L) with bound (R)-carvone (PDB: 4GWE) as model for carvone binding in normal orientation. (C) OYE1(W116I) with bound (S)-carvone (PDB: 4GE8) shows substrate in flipped orientation. The repositioning of the isopropenyl group near I116 eliminates the need for conformational changes of F296 and Y375. (D) Overlay of cpOYE303 holoenzyme (PDB: 4RNU) and cpOYE303 with bound HBA (PDB: 4RNV). Hydrogen bonding interactions and distances (in angstroms) are indicated.

Figure 5

Summary of catalytic activity and diastereoselectivity of OYE1, cpOYE154, and cpOYE303 as well as their respective W116I variants. The percent conversion of (S)-carvone (3) is indicated by column height, and diastereoselectivity based on formation of (R/S)-4 (blue) versus (S/S)-5 (yellow) is shown in the pie diagrams.