Anna Pabis

Promiscuity and electrostatic flexibility in the alkaline phosphatase superfamily

Catalytic promiscuity, that is, the ability of single enzymes to facilitate the turnover of multiple, chemically distinct substrates, is a widespread phenomenon that plays an important role in the evolution of enzyme function. Additionally, such pre-existing multifunctionality can be harnessed in artificial enzyme design. The members of the alkaline phosphatase superfamily have served extensively as both experimental and computational model systems for enhancing our understanding of catalytic promiscuity. In this Opinion, we present key recent computational studies into the catalytic activity of these highly promiscuous enzymes, highlighting the valuable insight they have provided into both the molecular basis for catalytic promiscuity in general, and its implications for the evolution of phosphatase activity.

Promiscuity in the Enzymatic Catalysis of Phosphate and Sulfate Transfer

The enzymes that facilitate phosphate and sulfate hydrolysis are among the most proficient natural catalysts known to date. Interestingly, a large number of these enzymes are promiscuous catalysts that exhibit both phosphatase and sulfatase activities in the same active site and, on top of that, have also been demonstrated to efficiently catalyze the hydrolysis of other additional substrates with varying degrees of efficiency. Understanding the factors that underlie such multifunctionality is crucial both for understanding functional evolution in enzyme superfamilies and for the development of artificial enzymes. In this Current Topic, we have primarily focused on the structural and mechanistic basis for catalytic promiscuity among enzymes that facilitate both phosphoryl and sulfuryl transfer in the same active site, while comparing this to how catalytic promiscuity manifests in other promiscuous phosphatases. We have also drawn on the large number of experimental and computational studies of selected model systems in the literature to explore the different features driving the catalytic promiscuity of such enzymes. Finally, on the basis of this comparative analysis, we probe the plausible origins and determinants of catalytic promiscuity in enzymes that catalyze phosphoryl and sulfuryl transfer.

Probing the mechanisms for the selectivity and promiscuity of methyl parathion hydrolase

Diverse organophosphate hydrolases have convergently evolved the ability to hydrolyse man-made organophosphates. Thus, these enzymes are attractive model systems for studying the factors shaping enzyme functional evolution. Methyl parathion hydrolase (MPH) is an enzyme from the metallo-β-lactamase superfamily, which hydrolyses a wide range of organophosphate, aryl ester and lactone substrates. In addition, MPH demonstrates metal-ion-dependent selectivity patterns. The origins of this remain unclear, but are linked to open questions about the more general role of metal ions in functional evolution and divergence within enzyme superfamilies. Here, we present detailed mechanistic studies of the paraoxonase and arylesterase activities of MPH complexed with five different transition metal ions, and demonstrate that the hydrolysis reactions proceed via similar pathways and transition states. However, while it is possible to discern a clear structural origin for the selectivity between different substrates, the selectivity between different metal ions appears to lie instead in the distinct electrostatic properties of the metal ions themselves, which causes subtle changes in transition state geometries and metal–metal distances at the transition state rather than significant structural changes in the active site. While subtle, these differences can be significant for shaping the metal-ion-dependent activity patterns observed for this enzyme. This article is part of the themed issue ‘Multiscale modelling at the physics–chemistry–biology interface’.

Cooperativity and flexibility in enzyme evolution

Enzymes are flexible catalysts, and there has been substantial discussion about the extent to which this flexibility contributes to their catalytic efficiency. What has been significantly less discussed is the extent to which this flexibility contributes to their evolvability. Despite this, recent years have seen an increasing number of both experimental and computational studies that demonstrate that cooperativity and flexibility play significant roles in enzyme innovation. This review covers key developments in the field that emphasize the importance of enzyme dynamics not just to the evolution of new enzyme function(s), but also as a property that can be harnessed in the design of new artificial enzymes.

Cryptic genetic variation defines the adaptive evolutionary potential of enzymes

Genetic variation among orthologous proteins can cause cryptic phenotypic properties that only manifest in changing environments. Such variation may also impact the evolutionary potential of proteins, but the molecular basis for this remains unclear. Here we perform comparative directed evolution in which four orthologous metallo-β-lactamases were evolved toward a new function. We found that genetic variation between these enzymes resulted in distinct evolutionary outcomes. The ortholog with the lower initial activity reached a 20-fold higher fitness plateau exclusively via increasing catalytic activity. By contrast, the ortholog with the highest initial activity evolved to a less-optimal and phenotypically distinct outcome through changes in expression, oligomerization and activity. We show that the cryptic molecular properties and conformational variation of residues in the initial genotypes cause epistasis, thereby constraining evolutionary outcomes. Our work highlights that understanding the molecular details relating genetic variation to protein functions is essential to predicting the evolution of proteins.

Extending the Nonbonded Cationic Dummy Model to Account for Ion-Induced Dipole Interactions

Modeling metalloproteins often requires classical molecular dynamics (MD) simulations in order to capture their relevant motions, which in turn necessitates reliable descriptions of the metal centers involved. One of the most successful approaches to date is provided by the “cationic dummy model”, where the positive charge of the metal ion is transferred toward dummy particles that are bonded to the central metal ion in a predefined coordination geometry. While this approach allows for ligand exchange, and captures the correct electrostatics as demonstrated for different divalent metal ions, current dummy models neglect ion-induced dipole interactions. In the present work, we resolve this weakness by taking advantage of the recently introduced 12–6–4 type Lennard-Jones potential to include ion-induced dipole interactions. We revise our previous dummy model for Mg2+ and demonstrate that the resulting model can simultaneously reproduce the experimental solvation free energy and metal–ligand distances without the need for artificial restraints or bonds. As ion-induced dipole interactions become particularly important for highly charged metal ions, we develop dummy models for the biologically relevant ions Al3+, Fe3+, and Cr3+. Finally, the effectiveness of our new models is demonstrated in MD simulations of several diverse (and highly challenging to simulate) metalloproteins.