Abstract
Members of enzyme superfamilies specialize in different reactions but often exhibit catalytic promiscuity for one another's reactions, consistent with catalytic promiscuity as an important driver in the evolution of new enzymes. Wanting to understand how catalytic promiscuity and other factors may influence evolution across a superfamily, we turned to the well-studied alkaline phosphatase (AP) superfamily, comparing three of its members, two evolutionarily distinct phosphatases and a phosphodiesterase. We mutated distinguishing active-site residues to generate enzymes that had a common Zn2+ bimetallo core but little sequence similarity and different auxiliary domains. We then tested the catalytic capabilities of these pruned enzymes with a series of substrates. A substantial rate enhancement of ∼1011-fold for both phosphate mono- and diester hydrolysis by each enzyme indicated that the Zn2+ bimetallo core is an effective mono/di-esterase generalist and that the bimetallo cores were not evolutionarily tuned to prefer their cognate reactions. In contrast, our pruned enzymes were ineffective sulfatases, and this limited promiscuity may have provided a driving force for founding the distinct one-metal-ion branch that contains all known AP superfamily sulfatases. Finally, our pruned enzymes exhibited 107-108-fold phosphotriesterase rate enhancements, despite absence of such enzymes within the AP superfamily. We speculate that the superfamily active-site architecture involved in nucleophile positioning prevents accommodation of the additional triester substituent. Overall, we suggest that catalytic promiscuity, and the ease or difficulty of remodeling and building onto existing protein scaffolds, have greatly influenced the course of enzyme evolution. Uncovering principles and properties of enzyme function, promiscuity, and repurposing provides lessons for engineering new enzymes.
Highlights
Members of enzyme superfamilies specialize in different reactions but often exhibit catalytic promiscuity for one another’s reactions, consistent with catalytic promiscuity as an important driver in the evolution of new enzymes
The AP superfamily subdivides into two main subgroups, one with enzymes containing a single metal ion and predominantly catalyzing sulfatase reactions and the other with enzymes containing the canonical alkaline phosphatase bimetallo center [38]
We focused on the latter group, which possesses two metal ions coordinated by conserved residues in the active site and a universally conserved serine or threonine nucleophile (Fig. 1)
Summary
Members of enzyme superfamilies specialize in different reactions but often exhibit catalytic promiscuity for one another’s reactions, consistent with catalytic promiscuity as an important driver in the evolution of new enzymes. Information about the physical and mechanistic features that determine catalytic efficiency and catalytic promiscuity will help us understand how enzymes have emerged throughout evolution and the evolutionary potential and evolvability of enzymes. These insights may be harnessed to engineer new, beneficial enzymes To ensure that our results would be representative rather than idiosyncratic, we compared three alkaline phosphatase (AP) superfamily members, mutating the distinguishing active-site residues to create mutant enzymes with a common Zn2ϩ bimetallo core but pervasive sequence differences and distinct auxiliary domains. Determination of the catalysis of these three “pruned” enzymes across a series of reaction classes and consideration of catalytic and structural features allowed us to relate intrinsic catalytic prowess of the Zn2ϩ bimetallo core to evolutionary decisions made across this superfamily
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