Structure-Guided Tuning of a Hydroxynitrile Lyase to Accept Rigid Pharmaco Aldehydes

Abstract

The chiral vicinal C–O/C–N bifunctional groups generated from enzymatic hydrocyanation represents a useful methodology. However, construction of the pharmacophore of β2-adrenoreceptor agonists with this method remains a great challenge because of complete racemization of the benzylic alcohol during deprotection of the acetal groups. In this study, structure-guided redesign of a hydroxynitrile lyase originating from Prunus communis (PcHNL5) enables a highly enantioselective hydrocyanation of rigid benzo-ketal aldehyde which was proved to be resistant against racemization during the deprotection step, with dramatically improved productivity (>95% conversion vs <1%). X-ray structure analysis and kinetic study revealed the side chain of L331 tunes the substrate adaptability of bulky and rigid benzo-ketal aldehyde, thereby facilitating the formation of a series of valuable unnatural cyanohydrins in high enantiopurities and good yields. Furthermore, the HNL variant L331A was successfully applied for a gram-scale chemo-enzymatic synthesis of (R)-salmeterol, a long-term β2-adrenoreceptor agonist, in an optically pure form (>99% ee) with an overall yield of 54%, which is the highest value reported.

Targeting Penicillium expansum GMC Oxidoreductase with High Affinity Small Molecules for Reducing Patulin Production

“…A pair of histidine residues (H564 and H608, P. expansum GMC oxidoreductase residues numbering) and two aromatic residues F563 and Y149 appeared to be crucial for the binding of patulin ligand in P. expansum GMC oxidoreductase. Notably, all the investigated GMC oxidoreductase homologous structures, crystallized in complex with ligands hosting aromatic functional groups, showed in their catalytic site always two histidine residues and one aromatic residue, forming an aromatic binding pocket (see i.e., ( R )-mandelonitrile lyase in complex with benzaldehyde, “6lqy.pdb”, [ 32 ]; pyridoxine 4-oxidase in complex with 4-(aminomethyl)-5-(hydroxymethyl)-2-methylpyridin-3-ol, “4ha6.pdb”, [ 30 ]; aryl-alcohol oxidase in complex with p -anisic acid, “5oc1.pdb”, [ 29 ]; ( R )-oxynitrile lyase isoenzyme 1 in complex with benzaldehyde, “3gdn.pdb”, [ 31 ]). Conversely, the fatty acid photodecarboxylase crystallized in complex with palmitate, “5ncc.pdb” [ 28 ] in correspondence of P. expansum GMC oxidoreductase H564, H608, F563 and Y149 hosted hydrophobic/hydrophilic (not aromatic) residues, i.e.…”

“…Similarly, considering that all the GMC oxidoreductases host a similarly located substrate binding region with two characteristic histidine residues (with the exception of 5ncc.pdb hosting a histidine and an arginine residue [ 28 ]), the 3D comparative model of the P. expansum GMC oxidoreductase was superimposed to the crystallized GMC oxidoreductases: “5oc1.pdb”, hosting p -anisic acid in the catalytic binding pocket, [ 29 ]; “4ha6.pdb”, hosting 4-(aminoethyl)-5-(hydroxymethyl)-2-methylpyridin-3-ol in its catalytic binding pocket, [ 30 ]; “5ncc.pdb”, hosting palmitic acid in its catalytic binding pocket [ 28 ]; “3gdn.pdb” and “6lqy.pdb”, hosting benzaldehyde in their catalytic binding pockets [ 31 , 32 ] for highlighting a protein region that contained all the cited crystallized superimposed ligands. The obtained protein region was supposed to be involved in the binding of patulin and/or its ( E )-ascladiol precursor and explored for investigating patulin/( E )-ascladiol binding modes and new P. expansum GMC oxidoreductase high affinity ligands to be tested in in vitro assays.…”

A High‐Throughput Screening Method for the Directed Evolution of Hydroxynitrile Lyase towards Cyanohydrin Synthesis

“…The freedom of applying structurally diverse aldehydes prompted us to perform a substrate screening of several well‐known HNLs to validate the current HTS methodology. We tested the enzymatic hydrocyanation reactions of reported ( S )‐ Me HNL H103L [13] and its variant H103L/W128A, [14] ( R )‐ Pc HNL5 [12] and its variant L331A, [15] and industrially applied ( R )‐ Pa HNL5 variants N3I/I108M/A111G [6c] and V317A [16] . Four aldehydes, α2 , α4 , α12 , and α25 , were used as representative substrates (Table 1).…”

Secretory Expression of Cyclohexanone Monooxygenase by Methylotrophic Yeast for Efficient Omeprazole Sulfide Bio-oxidation

“…Puri ed CHMO Acineto -Mut-E and CHMO Acineto -Mut-P was diluted with denature buffer to 1 mg/mL and incubated at 95°C for 10 min, then cooled to room temperature. 10 μg of the sample was mixed with 10 μg of the SpEndo H and incubated at 37°C for 1 h. Expression, puri cation of SpEndo H was conducted according to our previous work [Zheng et al 2020]. Samples were further analysed by SDS-PAGE.…”

“…One of the most important advantages of using methylotrophic yeast as an expression system for biotransformation is the direct use of yeast secretion as the catalyst [Zheng et al 2020;Qian et al 2014]. Bio-asymmetric synthesis of (S)-omeprazole by utilizing CHMO Acineto -Mut-P in the yeast secretion was then conducted.…”