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Title: Differential catalytic promiscuity of the alkaline phosphatase superfamily bimetallo core reveals mechanistic features underlying enzyme evolution

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. Here, we mutated distinguishing active-site residues to generate enzymes that had a common Zn 2+ 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 Zn 2+ 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 10 7–10 8-fold phosphotriesterase rate enhancements, despite absence of such enzymes within the AP superfamily.more » 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.« less
 [1] ;  [1] ;  [2] ;  [3] ;  [1] ;  [4]
  1. Stanford Univ., CA (United States). Dept. of Biochemistry, Beckman Center
  2. Stanford Univ., CA (United States). Dept. of Molecular and Cellular Physiology, Dept. of Neurology and Neurological Science, Structural Biology, and Photon Science, and Howard Hughes Medical Inst.
  3. SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL), Macromolecular Crystallographic Group
  4. Stanford Univ., CA (United States). Dept. of Biochemistry, Dept. of Neurology and Neurological Science, and Beckman Center; Stanford Univ., CA (United States). Stanford Chemistry, Engineering, and Medicine for Human Health (ChEM-H)
Publication Date:
Grant/Contract Number:
AC02-76SF00515; GM64798; GM049243
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 292; Journal Issue: 51; Journal ID: ISSN 0021-9258
American Society for Biochemistry and Molecular Biology
Research Org:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org:
USDOE; National Institutes of Health (NIH)
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES; enzyme; enzyme catalysis; evolution; phosphatase; substrate specificity
OSTI Identifier: