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Atopic dermatitis (AD) is characterized by a T-helper cell type 2 (Th2) inflammatory response leading to skin damage with erythema and edema. Comparative fecal sample analysis has uncovered a strong correlation between AD and Faecalibacterium prausnitzii strain A2-165, specifically associated with butyrate production. Therefore, understanding the functional mechanisms of crucial enzymes in the butyrate pathway, such as 3-hydroxybutyryl-CoA dehydrogenase of A2-165 (A2HBD), is imperative. Here, we have successfully elucidated the three-dimensional structure of A2HBD in complex with acetoacetyl-CoA and NAD + at a resolution of 2.2Å using the PAL-11C beamline (third generation). Additionally, X-ray data of A2HBD in complex with acetoacetyl-CoA at a resolution of 1.9 Å were collected at PAL-XFEL (fourth generation) utilizing Serial Femtosecond Crystallography (SFX). The monomeric structure of A2HBD consists of two domains, N-terminal and C-terminal, with cofactor binding occurring at the N-terminal domain, while the C-terminal domain facilitates dimerization. Our findings elucidate the binding mode of NAD + to A2HBD. Upon acetoacetyl-CoA binding, the crystal structure revealed a significant conformational change in the Clamp-roof domain (root-mean-square deviation of 2.202 Å). Notably, residue R143 plays a critical role in capturing the adenine phosphate ring, underlining its significance in substrate recognition and catalytic activity. The binding mode of acetoacetyl-CoA was also clarified, indicating its lower stability compared to NAD + . Furthermore, the conformational change of hydrophobic residues near the catalytic cavity upon substrate binding resulted in cavity shrinkage from an open to closed conformation. This study confirms the conformational changes of catalytic triads involved in the catalytic reaction and presents a proposed mechanism for substrate reduction based on structural observations.

Polyhydroxyalkanoates are gaining importance due to their biodegradable nature and close analogy to plastics. Polyhydroxybutyrate (PHB) is the most widely used bioplastic from polyalkanoate family, which is produced by a legion of bacterial species via phbCAB operon encoding β-ketothiolase (PhaA), NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) and polyhydroxyalkanoate synthase (PhaC). Augmentation in the activity of these enzymes is promising for increased PHB production which is achieved by enzyme engineering strategies including non-structural and structural approaches. Our study is deployed on directed evolution-based experimentally reported mutants of PhaB enzyme with increased efficiency due to impact on critical structural factors. We have analyzed and compared the native PhaB with two of its variants Q47L and T173S in complex with their cofactor i.e. NADPH as well as the substrate i.e. acetoacetyl-CoA, via long range molecular dynamics simulations. Interaction profile, MMPBSA, essential dynamics, and free energy landscape analysis revealed that the enzyme efficiency is critically affected by cofactor interactions. It was also observed that mutants have higher equilibrium constant with lesser but optimal affinity for substrate and cofactor than the wild type, which might be the reason for increased efficiency of the mutants via enhanced substrate and cofactor exchange rate. Our study provides insights into the cofactor and substrate binding affinities to PhaB enzyme at atomistic level, which will facilitate designing of highly efficient PhaB enzymes for increased PHB production. Communicated by Ramaswamy H. Sarma.