This review compiles recent structural insights to deepen our comprehension of the functional diversity of Fe/2OG DOs, highlighting the connection between enzyme structure and function in catalytic reactions for natural product synthesis.

Alcohol dehydrogenase (ADH) has attracted much attention due to its ability to catalyze the synthesis of important chiral alcohol pharmaceutical intermediates with high stereoselectivity. ADH protein engineering efforts have generally focused on reshaping the substrate-binding pocket. However, distant sites outside the pocket may also affect its activity, although the underlying molecular mechanism remains unclear. The current study aimed to apply evolutionary coupling-inspired engineering to the ADH CpRCR and to identify potential mutation sites. Through conservative analysis, phylogenic analysis and residues distribution analysis, the co-evolution hotspots Leu34 and Leu137 were confirmed to be highly evolved under the pressure of natural selection and to be possibly related to the catalytic function of the protein. Hence, Leu34 and Leu137, far away from the active center, were selected for mutation. The generated CpRCR-L34A and CpRCR-L137V variants showed high stereoselectivity and 1.24–7.81 fold increase in k_cat/K_m value compared with that of the wild type, when reacted with 8 aromatic ketones or β-ketoesters. Corresponding computational study implied that L34 and L137 may extend allosteric fluctuation in the protein structure from the distal mutational site to the active site. Moreover, the L34 and L137 mutations modified the pre-reaction state in multiple ways, in terms of position of the hydride with respect to the target carbonyl. These findings provide insights into the catalytic mechanism of the enzyme and facilitate its regulation from the perspective of the site interaction network.

Enzyme thermostabilization is considered a critical and often obligatory step in biosynthesis, because thermostability is a significant property of enzymes that can be used to evaluate their feasibility for industrial applications. However, conventional strategies for thermostabilizing enzymes generally introduce non-covalent interactions and/or natural covalent bonds caused by natural amino acid substitutions, and the trade-off between the activity and stability of enzymes remains a challenge. Here, we developed a computationally guided strategy for constructing thioether staples by incorporating noncanonical amino acid (ncAA) into the more flexible N/C-terminal domains of the multi-modular pullulanase from Bacillus thermoleovorans (BtPul) to enhance its thermostability. First, potential thioether staples located in the N/C-terminal domains of BtPul were predicted using RosettaMatch. Next, eight variants involving stable thioether staples were precisely predicted using FoldX and Rosetta ddg_monomer. Six positive variants were obtained, of which T73(O2beY)-171C had a 157% longer half-life at 70 °C and an increase of 7.0 °C in Tm, when compared with the wild-type (WT). T73(O2beY)-171C/T126F/A72R exhibited an even more improved thermostability, with a 211% increase in half-life at 70 °C and a 44% enhancement in enzyme activity compared with the WT, which was attributed to further optimization of the local interaction network. This work introduces and validates an efficient strategy for enhancing the thermostability and activity of multi-modular enzymes.

This review covers the recent progress in various biological approaches applied to the synthesis of enantiomerically pure cyclic and heterocyclic alcohols through CO/C–H/C–O asymmetric reactions.