Title: Structure of an ancestral mammalian family 1B1 cytochrome P450 with increased thermostability

Abstract

Mammalian cytochrome P450 enzymes often metabolize many pharmaceuticals and other xenobiotics, a feature that is valuable in a biotechnology setting. However, extant P450 enzymes are typically relatively unstable, with T₅₀ values of ~30–40 °C. Reconstructed ancestral cytochrome P450 enzymes tend to have variable substrate selectivity compared with related extant forms, but they also have higher thermostability and therefore may be excellent tools for commercial biosynthesis of important intermediates, final drug molecules, or drug metabolites. In this work, the mammalian ancestor of the cytochrome P450 1B subfamily was characterized structurally and functionally, revealing differences from the extant human CYP1B1 in ligand binding, metabolism, and potential molecular contributors to its thermostability. Whereas extant human CYP1B1 has one molecule of α-naphthoflavone in a closed active site, we observed that subtle amino acid substitutions outside the active site in the ancestor CYP1B enzyme yielded an open active site with four ligand copies. A structure of the ancestor with 17β-estradiol revealed only one molecule in the active site, which still had the same open conformation. Detailed comparisons between the extant and ancestor forms revealed increases in electrostatic and aromatic interactions between distinct secondary structure elements in the ancestral forms that may contribute to their thermostability.more » To the best of our knowledge, this represents the first structural evaluation of a reconstructed ancestral cytochrome P450, revealing key features that appear to contribute to its thermostability.« less

Authors:

Bart, Aaron G.  ^[1];

• Search DOE PAGES for author "Bart, Aaron G."
• Search DOE PAGES for ORCID "0000-0002-8605-4151"

Harris, Kurt L.  ^[2];

• Search DOE PAGES for author "Harris, Kurt L."
• Search DOE PAGES for ORCID "0000-0001-8301-3904"

Gillam, Elizabeth M. J.  ^[2];

• Search DOE PAGES for author "Gillam, Elizabeth M. J."
• Search DOE PAGES for ORCID "0000-0003-0378-793X"

Scott, Emily E.  ^[1]

• Search DOE PAGES for author "Scott, Emily E."
• Search DOE PAGES for ORCID "0000-0002-5538-5716"

---

+ Show Author Affiliations

1. Univ. of Michigan, Ann Arbor, MI (United States)
2. Univ. of Queensland St. Lucia, Brisbane, QLD (Australia)

Publication Date:

Tue Mar 10 00:00:00 EDT 2020

Research Org.:

Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS); SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)

Sponsoring Org.:

National Institutes of Health (NIH); USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institute of General Medical Sciences (NIGMS)

OSTI Identifier:

1657595

Grant/Contract Number:  

AC02-06CH11357; AC02-76SF00515; R37 GM076343

Resource Type:

Accepted Manuscript

Journal Name:

Journal of Biological Chemistry

Additional Journal Information:

Journal Volume: 295; Journal Issue: 17; Journal ID: ISSN 0021-9258

Publisher:

American Society for Biochemistry and Molecular Biology

Country of Publication:

United States

Language:

ENGLISH

Subject:

59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; cytochrome P450; synthetic biology; drug metabolism; enzyme structure; protein structure; membrane protein; X-ray crystallography; alpha-naphthoflavone; ancestral reconstruction; CYP1B1; estradiol; human; enzyme stability; thermostability; reconstructed ancestral protein; protein engineering; 16beta-estradiol