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Comparative Analysis and Ancestral Sequence Reconstruction of Bacterial Sortase Family Proteins Generates Functional Ancestral Mutants with Different Sequence Specificities

Valgardson, Jordan D. and Struyvenberg, Sarah A. and Sailer, Zachary R. and Piper, Isabel M. and Svendsen, Justin E. and Johnson, D. Alex and Vogel, Brandon A. and Antos, John M. and Harms, Michael J. and Amacher, Jeanine F. (2022) Comparative Analysis and Ancestral Sequence Reconstruction of Bacterial Sortase Family Proteins Generates Functional Ancestral Mutants with Different Sequence Specificities. Bacteria, 1 (2). pp. 121-135. ISSN 2674-1334. doi:10.3390/bacteria1020011.

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Gram-positive bacteria are some of the earliest known life forms, diverging from gram-negative bacteria 2 billion years ago. These organisms utilize sortase enzymes to attach proteins to their peptidoglycan cell wall, a structural feature that distinguishes the two types of bacteria. The transpeptidase activity of sortases make them an important tool in protein engineering applications, e.g., in sortase-mediated ligations or sortagging. However, due to relatively low catalytic efficiency, there are ongoing efforts to create better sortase variants for these uses. Here, we use bioinformatics tools, principal component analysis and ancestral sequence reconstruction, in combination with protein biochemistry, to analyze natural sequence variation in these enzymes. Principal component analysis on the sortase superfamily distinguishes previously described classes and identifies regions of relatively high sequence variation in structurally-conserved loops within each sortase family, including those near the active site. Using ancestral sequence reconstruction, we determined sequences of ancestral Staphylococcus and Streptococcus Class A sortase proteins. Enzyme assays revealed that the ancestral Streptococcus enzyme is relatively active and shares similar sequence variation with other Class A Streptococcus sortases. Taken together, we highlight how natural sequence variation can be utilized to investigate this important protein family, arguing that these and similar techniques may be used to discover or design sortases with increased catalytic efficiency and/or selectivity for sortase-mediated ligation experiments.

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Amacher, Jeanine F.0000-0002-3646-7521
Additional Information:© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( Received: 29 April 2022 / Revised: 25 May 2022 / Accepted: 7 June 2022 / Published: 9 June 2022. J.F.A. and J.M.A. were both funded by Cottrell Scholar Awards from the Research Corporation for Science Advancement. J.F.A. was also funded by NSF CHE-CAREER-2044958. M.J.H. was funded by NSF DEB-CAREER-1844963. In addition, I.M.P. received an Elwha Undergraduate Summer Research Award and D.A.J. received a Joseph & Karen Morse Student Research in Chemistry Fellowship to fund summer research. Author Contributions. Conceptualization, J.D.V., Z.R.S., M.J.H. and J.F.A.; methodology, J.D.V., Z.R.S., M.J.H. and J.F.A.; software, J.D.V., Z.R.S. and M.J.H.; validation, S.A.S., I.M.P., J.E.S., D.A.J. and B.A.V.; formal analysis, J.D.V., J.M.A. and J.F.A.; investigation, J.D.V., S.A.S., Z.R.S., I.M.P., J.E.S., D.A.J. and B.A.V.; resources, J.M.A., M.J.H. and J.F.A.; data curation, J.D.V. and J.F.A.; writing—original draft preparation, J.F.A.; writing—review and editing, J.D.V., S.A.S., I.M.P., J.E.S., J.M.A., M.J.H. and J.F.A.; visualization, J.D.V. and J.F.A.; supervision, J.M.A., M.J.H. and J.F.A.; project administration, J.F.A.; funding acquisition, J.M.A., M.J.H. and J.F.A. All authors have read and agreed to the published version of the manuscript. Supplementary Materials. The following supporting information can be downloaded at:, Figure S1: Principal Component Analysis (PCA) of sortase superfamily; Figure S2: Structures of spSrtA from the AlphaFold database and homology modeling; Figure S3: Size exclusion chromatography of ancestral SrtA proteins; Recombinant protein sequences used in this study [14,25]. The authors declare no conflict of interest.
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Cottrell Scholar of Research CorporationUNSPECIFIED
Western Washington UniversityUNSPECIFIED
Subject Keywords:sortases; enzymes; protein engineering; principal component analysis; network analysis; bioinformatics; ancestral sequence reconstruction; evolution
Issue or Number:2
Record Number:CaltechAUTHORS:20220722-769578000
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Official Citation:Valgardson, Jordan D., Sarah A. Struyvenberg, Zachary R. Sailer, Isabel M. Piper, Justin E. Svendsen, D. Alex Johnson, Brandon A. Vogel, John M. Antos, Michael J. Harms, and Jeanine F. Amacher. 2022. "Comparative Analysis and Ancestral Sequence Reconstruction of Bacterial Sortase Family Proteins Generates Functional Ancestral Mutants with Different Sequence Specificities" Bacteria 1, no. 2: 121-135.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:115800
Deposited By: George Porter
Deposited On:26 Jul 2022 17:34
Last Modified:26 Jul 2022 17:34

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