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      When directed evolution met ancestral enzyme resurrection

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      1 ,
      Microbial Biotechnology
      John Wiley and Sons Inc.

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          Abstract

          The directed evolution of ancestral ‐resurrected‐ enzymes can give a new twist in protein engineering approaches towards more versatile and robust biocatalysts.

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          In the light of directed evolution: pathways of adaptive protein evolution.

          Directed evolution is a widely-used engineering strategy for improving the stabilities or biochemical functions of proteins by repeated rounds of mutation and selection. These experiments offer empirical lessons about how proteins evolve in the face of clearly-defined laboratory selection pressures. Directed evolution has revealed that single amino acid mutations can enhance properties such as catalytic activity or stability and that adaptation can often occur through pathways consisting of sequential beneficial mutations. When there are no single mutations that improve a particular protein property experiments always find a wealth of mutations that are neutral with respect to the laboratory-defined measure of fitness. These neutral mutations can open new adaptive pathways by at least 2 different mechanisms. Functionally-neutral mutations can enhance a protein's stability, thereby increasing its tolerance for subsequent functionally beneficial but destabilizing mutations. They can also lead to changes in "promiscuous" functions that are not currently under selective pressure, but can subsequently become the starting points for the adaptive evolution of new functions. These lessons about the coupling between adaptive and neutral protein evolution in the laboratory offer insight into the evolution of proteins in nature.
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            Hyperstability and substrate promiscuity in laboratory resurrections of Precambrian β-lactamases.

            We report a sequence reconstruction analysis targeting several Precambrian nodes in the evolution of class-A β-lactamases and the preparation and experimental characterization of their encoded proteins. Despite extensive sequence differences with the modern enzymes (~100 amino acid differences), the proteins resurrected in the laboratory properly fold into the canonical lactamase structure. The encoded proteins from 2-3 billion years (Gyr)-old β-lactamase sequences undergo cooperative two-state thermal denaturation and display very large denaturation temperature enhancements (~35 °C) relative to modern β-lactamases. They degrade different antibiotics in vitro with catalytic efficiencies comparable to that of an average modern enzyme. This enhanced substrate promiscuity is not accompanied by significant changes in the active-site region as seen in static X-ray structures, suggesting a plausible role for dynamics in the evolution of function in these proteins. Laboratory resurrections of 2-3 Gyr-old β-lactamases also endowed modern microorganisms with significant levels of resistance toward a variety of antibiotics, opening up the possibility of performing laboratory replays of the molecular tape of lactamase evolution. Overall, these results support the notions that Precambrian life was thermophilic and that proteins can evolve from substrate-promiscuous generalists into specialists during the course of natural evolution. They also highlight the biotechnological potential of laboratory resurrection of Precambrian proteins, as both high stability and enhanced promiscuity (likely contributors to high evolvability) are advantageous features in protein scaffolds for molecular design and laboratory evolution.
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              Single-molecule paleoenzymology probes the chemistry of resurrected enzymes

              A journey back in time is possible at the molecular level by reconstructing proteins from extinct organisms. Here we report the reconstruction, based on sequence predicted by phylogenetic analysis, of seven Precambrian thioredoxin enzymes (Trx), dating back between ~1.4 and ~4 billion years (Gyr). The reconstructed enzymes are up to 32° C more stable than modern enzymes and the oldest show significantly higher activity than extant ones at pH 5. We probed their mechanisms of reduction using single-molecule force spectroscopy. From the force-dependency of the rate of reduction of an engineered substrate, we conclude that ancient Trxs utilize chemical mechanisms of reduction similar to those of modern enzymes. While Trx enzymes have maintained their reductase chemistry unchanged, they have adapted over a 4 Gyr time span to the changes in temperature and ocean acidity that characterize the evolution of the global environment from ancient to modern Earth.
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                Author and article information

                Contributors
                malcalde@icp.csic.es
                Journal
                Microb Biotechnol
                Microb Biotechnol
                10.1111/(ISSN)1751-7915
                MBT2
                Microbial Biotechnology
                John Wiley and Sons Inc. (Hoboken )
                1751-7915
                11 November 2016
                January 2017
                : 10
                : 1 ( doiID: 10.1111/mbt2.2017.10.issue-1 )
                : 22-24
                Affiliations
                [ 1 ] Department of Biocatalysis Institute of CatalysisCSIC Cantoblanco 28049 MadridSpain
                Author notes
                [*] [* ]For correspondence. E‐mail malcalde@ 123456icp.csic.es ; Tel. +34 91 5854806; Fax +34 91 5854760.
                Article
                MBT212452
                10.1111/1751-7915.12452
                5270717
                27863072
                246f23d8-5f64-41da-96b8-54f775c99a7f
                © 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

                This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 07 October 2016
                : 09 October 2016
                Page count
                Figures: 1, Tables: 0, Pages: 3, Words: 1363
                Categories
                Crystal Ball
                Crystal Ball
                Custom metadata
                2.0
                mbt212452
                January 2017
                Converter:WILEY_ML3GV2_TO_NLMPMC version:5.0.3 mode:remove_FC converted:27.01.2017

                Biotechnology
                Biotechnology

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