Enhancing the specific activity of 3α-hydroxysteroid dehydrogenase through cross-regional combinatorial mutagenesis.

3α-Hydroxysteroid dehydrogenase (3α-HSD) from Comamonas testosteroni is widely used in clinical settings to measure serum total bile acid levels. However, its low enzymatic activity leads to high operational costs. In this study, we employed a combinatorial mutagenesis approach to systematically identify potential key mutation sites within the enzyme.

Reshaping the QM Region On-the-Fly: Adaptive-Shape QM/MM Dynamic Simulations of a Hydrated Proton in Bulk Water.

Adaptive quantum mechanics/molecular mechanics (QM/MM) reclassifies on-the-fly a molecule or molecular fragment as QM or MM during dynamics simulations without abrupt changes in the energy or forces. Notably, the permuted adaptive-partitioning (PAP) algorithms have been applied to simulate a hydrated proton, with a mobile QM zone anchored at a pseudoatom called a proton indicator. The position of the proton indicator approximates the location of the delocalized excess proton, yielding a smooth trajectory of the proton diffusing via the Grotthuss mechanism in aqueous solutions.

Unraveling the Valence State and Reactivity of Copper Centers in Membrane-Bound Particulate Methane Monooxygenase.

Particulate methane monooxygenase (pMMO) plays a critical role in catalyzing the conversion of methane to methanol, constituting the initial step in the C1 metabolic pathway within methanotrophic bacteria. However, the membrane-bound pMMO's structure and catalytic mechanism, notably the copper's valence state and genuine active site for methane oxidation, have remained elusive. Based on the recently characterized structure of membrane-bound pMMO, extensive computational studies were conducted to address these long-standing issues.

Evaluating the Transition State Stabilization/Destabilization Effects of the Electric Fields from Scaffold Residues by a QM/MM Approach.

The protein scaffolds of enzymes not only provide structural support for the catalytic center but also exert preorganized electric fields for electrostatic catalysis. In recent years, uniform oriented external electric fields (OEEFs) have been widely applied to enzymatic reactions to mimic the electrostatic effects of the environment. However, the electric fields exerted by individual residues in proteins may be quite heterogeneous across the active site, with varying directions and strengths at different positions of the active site.

Tracking the Delocalized Proton in Concerted Proton Transfer in Bulk Water.

A solvated proton in water is often characterized as a charge or structural defect, and it is important to track its evolution on-the-fly in certain dynamics simulations. Previously, we introduced the proton indicator, a pseudo-atom, whose position approximates the location of the excess proton modeled as a structural defect. The proton indicator generally yields a smooth trajectory of a hydrated proton diffusing in aqueous solutions, including in the events of stepwise proton transfer via the Grotthuss mechanism; however, the proton indicator did not perform well in the events of concerted proton transfer, for which it occasionally yielded large position displacements between two successive time steps.

How Do Preorganized Electric Fields Function in Catalytic Cycles? The Case of the Enzyme Tyrosine Hydroxylase.

Nature has devised intrinsic electric fields (IEFs) that are engaged in electrostatic catalysis of enzymes. But, how does the IEF target its function in enzymes that involve several reaction steps in catalytic cycles? To decipher the impact of the IEF on the catalytic cycle of an enzyme system, we have performed molecular dynamics and quantum-mechanical/molecular-mechanical (QM/MM) simulations on tyrosine hydroxylase (TyrH). The catalytic cycle of TyrH involves two reaction stages: the activation of HO to form the active species of compound I (Cpd I), in the first stage, and the Cpd I-mediated hydroxylation of l-tyrosine to l-DOPA, in the second stage.

Molecular Mechanism of the Mononuclear Copper Complex-Catalyzed Water Oxidation from Cluster-Continuum Model Calculations.

Cluster-continuum model calculations were conducted to decipher the mechanism of water oxidation catalyzed by a mononuclear copper complex. Among various O-O bond formation mechanisms investigated in this study, the most favorable pathway involved the nucleophilic attack of OH onto the L-Cu -OH intermediate. During such process, the initial binding of OH to the proximity of L-Cu -OH would result in the spontaneous oxidation of OH , leading to OH⋅ radical and Cu -OH species.