Abstract
It is becoming widely accepted that catalytic promiscuity, i.e., the ability of a single enzyme to catalyze the turnover of multiple, chemically distinct substrates, plays a key role in the evolution of new enzyme functions. In this context, the members of the alkaline phosphatase superfamily have been extensively studied as model systems in order to understand the phenomenon of enzyme multifunctionality. In the present work, we model the selectivity of two multiply promiscuous members of this superfamily, namely the phosphonate monoester hydrolases from Burkholderia caryophylli and Rhizobium leguminosarum. We have performed extensive simulations of the enzymatic reaction of both wild-type enzymes and several experimentally characterized mutants. Our computational models are in agreement with key experimental observables, such as the observed activities of the wild-type enzymes, qualitative interpretations of experimental pH-rate profiles, and activity trends among several active site mutants. In all cases the substrates of interest bind to the enzyme in similar conformations, with largely unperturbed transition states from their corresponding analogues in aqueous solution. Examination of transition-state geometries and the contribution of individual residues to the calculated activation barriers suggest that the broad promiscuity of these enzymes arises from cooperative electrostatic interactions in the active site, allowing each enzyme to adapt to the electrostatic needs of different substrates. By comparing the structural and electrostatic features of several alkaline phosphatases, we suggest that this phenomenon is a generalized feature driving selectivity and promiscuity within this superfamily and can be in turn used for artificial enzyme design.
Highlights
We model the selectivity of two multiply promiscuous members of this superfamily, namely the phosphonate monoester hydrolases from Burkholderia caryophylli and Rhizobium leguminosarum
We demonstrate that despite their broad promiscuity, both phosphonate monoester hydrolases (PMHs) studied in this work hydrolyze all five chemically distinct substrates through a unified mechanism, binding substrates in similar positions and without the need for any significant local or global conformational changes
We demonstrate that the apparent resilience of these enzymes to active site mutations as well as the overall promiscuity is due to compensatory electrostatic effects from different residues, allowing enough flexibility in the electrostatic environment of the active site to accommodate multiple substrates with distinct transition states and charge distributions
Summary
In recent years,[1] it has become widely accepted that catalytic promiscuity, i.e., the ability of many enzymes to catalyze the turnover of multiple chemically distinct substrates, plays a key role in the evolution of new functions, allowing for rapid responses to environmental changes.[2,3] interest in this phenomenon has exploded as it has been increasingly shown to be a powerful tool for gaining knowledge not just into the process of natural functional evolution,[2] and as a factor that can be exploited in effective artificial enzyme design.[1,3,4] Such promiscuity appears to be highly pronounced among many phosphotransferases, such as the recently evolved bacterial phosphotriesterase (PTE),[5] serum paraoxonase 1 (PON1),[6] and members of the alkaline phosphatase (AP) superfamily,[7−9] to name a few examples. A particular hallmark of this superfamily is crosswise-promiscuity, in that the native substrate for one member of the superfamily is often a promiscuous substrate for another,[11,15] in some cases with high (and almost comparable) proficiencies toward both the native and promiscuous substrates.[8,9,12] As a result, these
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