Owing to their outstanding catalytic properties, enzymes represent powerful tools for carrying out a wide range of (bio)chemical transformations with high proficiency. In this context, enzymes with high biocatalytic promiscuity are somewhat neglected. Here, we demonstrate that a meticulous modification of a synthetic shell that surrounds an immobilized enzyme possessing broad substrate specificity allows the resulting nanobiocatalyst to be endowed with enantioselective properties while maintaining a high level of substrate promiscuity. Our results show that control of the enzyme nano-environment enables tuning of both substrate specificity and enantioselectivity. Further, we demonstrate that our strategy of enzyme supramolecular engineering allows the enzyme to be endowed with markedly enhanced stability in an organic solvent (i.e., acetonitrile). The versatility of the method was assessed with two additional substrate-promiscuous and structurally different enzymes, for which improvements in enantioselectivity and stability were confirmed. We expect this method to promote the use of supramolecularly engineered promiscuous enzymes in industrially relevant biocatalytic processes.
Glycosylation of polyphenols may increase their aqueous solubility, stability, bioavailability and pharmacological activity. Herein, we used a mutant of sucrose phosphorylase from Thermoanaerobacterium thermosaccharolyticum engineered to accept large polyphenols (variant TtSPP_R134A) to produce phloretin glucosides. The reaction was performed using 10% (v/v) acetone as cosolvent. The selective formation of a monoglucoside or a diglucoside (53% and 73% maximum conversion percentage, respectively) can be kinetically controlled. MS and 2D‐NMR determined that the monoglucoside was phloretin 4’‐O‐α‐D‐glucopyranoside and the diglucoside phloretin‐4’‐O‐[α‐D‐glucopyranosyl‐(1→3)‐O‐α‐D‐glucopyranoside], a novel compound. The molecular features that determine the specificity of this enzyme for 4’‐OH phenolic group were analysed by induced‐fit docking analysis of each putative derivative, using the crystal structure of TtSPP and changing the mutated residue. The mono‐ and diglucoside were, respectively, 71‐ and 1200‐fold more soluble in water than phloretin at room temperature. The α‐glucosylation decreased the antioxidant capacity of phloretin, measured by DPPH and ABTS assays; however, this loss was moderate and the activity could be recovered upon deglycosylation in vivo. Since phloretin attracts a great interest in dermocosmetic applications, we analyzed the percutaneous absorption of glucosides and the aglycon employing a pig skin model. Although the three compounds were detected in all skin layers (except the fluid receptor), the diglucoside was present mainly on superficial layers.
The yeast diadenosine and diphosphoinositol polyphosphate phosphohydrolase DDP1 is a Nudix enzyme with pyrophosphatase activity on diphosphoinositides, dinucleotides, and polyphosphates. These substrates bind to diverse protein targets and participate in signaling and metabolism, being essential for energy and phosphate homeostasis, ATPase pump regulation, or protein phosphorylation. An exhaustive structural study of DDP1 in complex with multiple ligands related to its three diverse substrate classes is reported. This allowed full characterization of the DDP1 active site depicting the molecular basis for endowing multisubstrate abilities to a Nudix enzyme, driven by phosphate anchoring following a defined path. This study, combined with multiple enzyme variants, reveals the different substrate binding modes, preferences, and selection. Our findings expand current knowledge on this important structural superfamily with implications extending beyond inositide research. This work represents a valuable tool for inhibitor/substrate design for ScDDP1 and orthologs as potential targets to address fungal infections and other health concerns.
The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for the ω‐1 hydroxylation of fatty acids with quenched stepwise over‐oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward subterminal hydroxylation with a drop in the over‐oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g L−1 in a fed‐batch bioreactor and used in the preparative synthesis of 1.4 g of (ω‐1)‐hydroxytetradecanoic acid with 95 % regioselectivity and 83 % ee for the S enantiomer.
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