Enzyme-catalyzed reactions have begun to transform pharmaceutical manufacturing, offering levels of selectivity and tunability that can dramatically improve chemical synthesis. Combining enzymatic reactions into multistep biocatalytic cascades brings additional benefits. Cascades avoid the waste generated by purification of intermediates. They also allow reactions to be linked together to overcome an unfavorable equilibrium or avoid the accumulation of unstable or inhibitory intermediates. We report an in vitro biocatalytic cascade synthesis of the investigational HIV treatment islatravir. Five enzymes were engineered through directed evolution to act on non-natural substrates. These were combined with four auxiliary enzymes to construct islatravir from simple building blocks in a three-step biocatalytic cascade. The overall synthesis requires fewer than half the number of steps of the previously reported routes.

Copper chaperones deliver reduced copper (i.e., Cu(I)) to metal-binding domains of P-type ATPases in the cytoplasm of a range of organisms. Both chaperones and target domains have a ferredoxin-like fold and metal-binding motifs involving two Cys residues. Here, we investigated the Cu-binding geometry and structural dynamics of two homologous Cu(I) chaperones, Homo sapiens Atox1 and Bacillus subtilis CopZ, using a combination of quantum mechanical-molecular mechanics (QM-MM) and classical molecular dynamics (MD) methods. Our QM-MM optimized geometries for the holo- proteins suggested that Cu(I) in Atox1 favors a linear Cys(S)-Cu-Cys(S) arrangement but that this angle is close to 150 degrees in CopZ. Classical MD simulations suggest that both Atox1 and CopZ apo- forms have an increased conformational flexibility as compared to the respective holo- forms. This difference is most pronounced in CopZ and correlates with a lower in vitro thermal stability. Both average fluctuation (i.e., rmsd) and radius of gyration data demonstrate that the effects of Cu(I) coordination extend throughout the proteins. Distinct deviations between the two homologues were found in protein-solvent interactions, entropy of Cu(I) binding, and apo-protein Cys-Cys distance distributions. Our in silico results provide new insights into copper chaperone behavior with direct implications for copper transport mechanisms in vivo.

ATP7A/B are human P(1B)-type ATPases involved in cellular Cu homeostasis. The N-terminal parts of these multidomain proteins contain six metal-binding domains (MBDs) connected by linkers. The MBDs are similar in structure to each other and to the human copper chaperone Atox1, although their distinct roles in Cu transfer appear to vary. All domains have the ferredoxin-like fold and a solvent-exposed loop with a MXCXXC motif that can bind Cu(I). Here, we investigated the dynamic behavior of the individual MBDs (WD1-WD6) in ATP7B in apo forms using molecular dynamic simulations. We also performed simulations of three Cu-bound forms (WD2c, WD4c, and WD6c). Our results reveal molecular features that vary distinctly among the MBDs. Whereas WD1, WD2, and WD6 have well-defined Cu loop conformations stabilized by a network of interactions, WD4 and WD5 exhibit greater loop flexibility and, in WD4, helix alpha1 unwinds and rewinds. WD3, which has the lowest sequence identity, behaves differently and its Cu loop is rigid with respect to the rest of the domain. Cu coordination reduces structural dynamics in all domains but WD4c. In agreement with predictions on individual domains, simulations of the six possible Atox1-WD heterocomplexes show that Atox1 interactions with WD4 are the strongest. This study provides molecular explanations for reported Cu transfer and protein-protein interaction specificity.

The molecular details of how copper (Cu) is transferred from the human Cu chaperone Atox1 to metal-binding domains (MBDs) of P1B-type ATPases are still unclear. Here, we use a computational approach, employing quantum mechanics/molecular mechanics (QM/MM) methods, to shed light on the reaction mechanism [probable intermediates, Cu(I) coordination geometries, activation barriers, and energetics] of Cu(I) transfer from Atox1 to the fourth MBD of Wilson disease protein (WD4). Both Atox1 and WD4 have solvent-exposed metal-binding motifs with two Cys residues that coordinate Cu(I). After assessing the existence of all possible 2-, 3- and 4-coordinate Cu-intermediate species, one dominant reaction path emerged. First, without activation barrier, WD4’s Cys1 binds Cu(I) in Atox1 to form a 3-coordinated intermediate. Next, with an activation barrier of about 9.5 kcal/mol, a second 3-coordinated intermediate forms that involves both of the Cys residues in WD4 and Cys1 of Atox1. This species can then form the product by decoordination of Atox1’s Cys1 (barrier of about 8 kcal/mol). Overall, the Cu-transfer reaction from Atox1 to WD4 appears to be kinetically accessible but less energetically favorable (ΔE = 7.7 kcal/mol). Our results provide unique insights into the molecular mechanism of protein-mediated Cu(I) transfer in the secretory pathway and are in agreement with existing experimental data.