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      Uncovering protein–protein interactions through a team-based undergraduate biochemistry course

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          Abstract

          How can we provide fertile ground for students to simultaneously explore a breadth of foundational knowledge, develop cross-disciplinary problem-solving skills, gain resiliency, and learn to work as a member of a team? One way is to integrate original research in the context of an undergraduate biochemistry course. In this Community Page, we discuss the development and execution of an interdisciplinary and cross-departmental undergraduate biochemistry laboratory course. We present a template for how a similar course can be replicated at other institutions and provide pedagogical and research results from a sample module in which we challenged our students to study the binding interface between 2 important biosynthetic proteins. Finally, we address the community and invite others to join us in making a larger impact on undergraduate education and the field of biochemistry by coordinating efforts to integrate research and teaching across campuses.

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          Integrating Teaching and Research in Undergraduate Biology Laboratory Education

          A course recently designed and implemented at Stanford University applies practical suggestions for creating research-based undergraduate courses that benefit both teaching and research.
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            Cytochrome p450sky interacts directly with the nonribosomal peptide synthetase to generate three amino acid precursors in skyllamycin biosynthesis.

            The generation of modified amino acid precursors for incorporation in nonribosomal peptide synthesis (NRPS) plays a crucial, if often understated, role in the generation of peptide natural products. The biosynthesis of the cyclic depsipeptide skyllamycin requires three β-hydroxylated amino acid precursors, with in vivo gene inactivation experiments implicating cytochrome P450sky (CYP163B3) in the hydroxylation of these amino acids. Here, we demonstrate the in vitro oxidation of l-amino acid substrates bound to peptidyl carrier protein (PCP) domains 5, 7, and 11 of the skyllamycin nonribosomal synthetase by P450sky. Selectivity for these domains over other PCP domains could be demonstrated, with hydroxylation selective for l-amino acids and stereospecific in nature resulting in the (2S,3S)-configuration. The oxidation of amino acids or small molecule substrate analogues was not supported, demonstrating the necessity of the carrier protein in P450sky-catalyzed hydroxylation. The binding of aminoacyl-PCP substrates to P450sky was detected for the catalytically active PCP7 but not for the catalytically inactive PCP10, indicating carrier protein-mediated selectivity in P450sky substrate binding. X-ray crystal structures of P450sky reveal a 3D-structure with a highly open active site, the size of which is dictated by the carrier protein bound nature of the substrate. P450sky is the first P450 demonstrated to not only interact directly with PCP-bound amino acids within the peptide-forming NRPS but also to do so with three different PCP domains in a specific fashion. This represents an expansion of the complexity and scope of NRPS-mediated peptide synthesis, with the generation of hydroxylated amino acid precursors occurring through the interaction of P450 enzymes following, rather than prior to, the selection of amino acids by NRPS-adenylation domains.
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              The structure of a transient complex of a nonribosomal peptide synthetase and a cytochrome P450 monooxygenase.

              Studying the interplay between nonribosomal peptide synthetases (NRPS), a major source of secondary metabolites, and crucial external modifying enzymes is a challenging task since the interactions involved are often transient in nature. By applying a range of synthetic inhibitor-type compounds, a stabilized complex appropriate for structural analysis was generated for such a tailoring enzyme and an NRPS domain. The complex studied comprises an NRPS peptidyl carrier protein (PCP) domain bound to the Cytochrome P450 enzyme that is crucial for the provision of β-hydroxylated amino acid precursors in the biosynthesis of the cyclic depsipeptide skyllamycin. The structure reveals that complex formation is governed by hydrophobic interactions, the presence of which can be controlled through minor alterations in PCP structure that enable selectivity amongst multiple highly similar PCP domains.
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                Author and article information

                Journal
                PLoS Biol
                PLoS Biol
                plos
                plosbiol
                PLoS Biology
                Public Library of Science (San Francisco, CA USA )
                1544-9173
                1545-7885
                1 November 2017
                November 2017
                1 November 2017
                : 15
                : 11
                : e2003145
                Affiliations
                [1 ] Department of Chemistry, Haverford College, Haverford, Pennsylvania, United States of America
                [2 ] Department of Biology, Haverford College, Haverford, Pennsylvania, United States of America
                [3 ] The Monash Biomedical Discovery Institute, EMBL Australia, Monash University, Clayton, Victoria, Australia
                [4 ] The Department of Biochemistry and Molecular Biology and ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria, Australia
                Author notes

                The authors have declared that no competing interests exist.

                ‡ Undergraduate student enrolled in 2015 Biochemistry 390 (“Biochemistry Superlab”)

                Author information
                http://orcid.org/0000-0002-4342-2815
                Article
                pbio.2003145
                10.1371/journal.pbio.2003145
                5683658
                29091712
                1c2c8891-d4db-4139-9f93-ab333200cbac
                © 2017 Cookmeyer et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                Page count
                Figures: 4, Tables: 0, Pages: 13
                Funding
                National Institutes of Health www.nih.gov (grant number R15GM120704). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. National Science Foundation www.nsf.gov (grant number CHE-1652424). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. National Science Foundation www.nsf.gov (grant number MCB-1243656). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Research Corporation for Scientific Advancement http://rescorp.org (grant number 23251). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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